Synthetic methods for aplidine and new antitumoral derivatives, methods of making and using them

ABSTRACT

The invention relates to aplidine derivatives of the general formula:  
                 
which are useful for the treatment of tumors.

The present invention relates to synthetic methods for aplidine and newantitumoral derivatives, methods of making and using them.

BACKGROUND OF THE INVENTION

Aplidine has a cyclic structure with a sidechain, as follows:

The didemnins form a class of cyclic depsipeptides which have beenisolated from various species of the Tndidemnum genus (Rinehart, Jr. etal. J. Am. Chem. Soc, 103, 1857-59 (1981), Rinehart, Jr. et al. Science,212, 933-935 (1981) with potent antitumoral and antiviral activities.Among them, aplidine is one of the most antitumoral active naturaldidemnins. Description of the isolation and antitumoral activity ofAplidine is provided in U.S. Pat. No. 5,834,586.

A number of synthetic or natural analogs of Aplidine have been described(Rinehart, Jr. et al. J. Med. Chem., 1996, 39, 2819-2834) that includedifferent modifications in the side chain, but preserving the samemacrocyclic structure.

Recently have been described a related structure of didemnins calledTamandarins (Fenical, W. et al., J. Org. Chem., 2000, 65, 782-792) whichwere isolated from an unidentified ascidian of the family didemnidae.These molecules were found to differ only by the presence ofhydroxyisovaleric acid (Hiv³), instead of the hydroxyisovalerylpropionicacid (Hip³). They have been described as highly active antiviral,antitumor and immunosuppresive peptides.

SUMMARY OF THE INVENTION

The present invention relates to the compounds described herein, termedaplidine derivatives for use in medicine, in particular in the treatmentof tumours. The invention also relates to pharmaceutical preparationscomprising them for treatment of tumours, for example, solid tumours,and use of the compound in the preparation of a medicament for thetreatment of tumours. Treatment of solid tumours such as bladder,breast, colon, gastric, liver, nscl, ovary, pancreas, pharynx, prostate,renal, scl, retinoblastoma, melanoma, fibrosarcoma, chondrosarcoma, orosteosarcoma, or treatment of leukemia/lymphomas such as ALL(Promyelocytic leukemia), ALL (Acute lymphobalstic), CML (Chronicmyelogenous), ALL (B-cell), leukemia (Hairy B-cell), leukemia (plasmacell), lymphoma (T cell), lymphoma (cutaneous T cell), lymphoma(undifferentiated), lymphoma (Burkitts B cell), lymphoma (histiocytic),lymphoma (B cell), lymphoma (Burkitts ascites) is particularlypreferred.

Examples of pharmaceutical compositions of the invention include anysolid (for example tablets, pils, capsules, granules) or liquid(solutions, suspensions or emulsions) with suitable composition or oral,topical or parenteral administration, and they may contain the purecompound or in combination with any carrier or other pharmacologicallyactive compounds. These compositions may need to be sterile whenadministered parenterally.

Suitably, the compound may be conjugated to a carrier protein or anothersuitable agent for delivery into the animal or human body. Conjugationmay occur directly between a carrier and the compound, or indirectly viaa suitable linker.

Administration of the compound or compositions of the present inventionmay be by any suitable method, such as intravenous infusion, oralpreparations, intraperitoneal and intravenous administration. We preferthat infusion times of up to 24 hours are used, more preferably 2-12hours, with 2-6 hours most preferred. Short infusion times which allowtreatment to be carried out without an overnight stay in hospital areespecially desirable. However, infusion may be 12 to 24 hours or evenlonger if required. Infusion may be carried out at suitable intervals ofsay 2 to 4 weeks. Pharmaceutical compositions containing compounds ofthe invention may be delivered by liposome or nanosphere encapsulation,in sustained release formulations or by other standard delivery means.

The correct dosage of the compound will vary according to the particularformulation, the mode of application, and the particular situs, host andcancer or tumour being treated. Other factors like age, body weight,sex, diet, time of administration, rate of excretion, condition of thehost, drug combinations, reaction sensitivities and severity of thedisease shall be taken into account. Administration can be carried outcontinuously or periodically within the maximum tolerated dose.

The compounds of this invention may be used with other drugs to providea combination therapy. The other drugs may form part of the samecomposition, or be provided as a separate composition for administrationat the same time or a different time. The identity of the other drug isnot particularly limited, and suitable candidates include:

-   a) drugs with antimitotic effects, especially those which target    cytoskeletal elements. including microtubule modulators such as    taxane drugs (such as taxol, paclitaxel, taxotere, docetaxel),    podophylotoxins or vinca alkaloids (vincristine, vinblastine);-   b) antimetabolite drugs such as 5-fluorouracil, cytarabine,    gemcitabine, purine analogues such as pentostatin, methotrexate);-   c) alkylating agents such as nitrogen mustards (such as    cyclophosphamide or ifosphamide);-   d) drugs which target DNA such as the antracycline drugs    adrian-iycin, doxorubicin, pharmorubicin or epirubicin;-   e) drugs which target such as etoposide; hormones and hormone    agonists or antagonists such as estrogens, antiestrogens (tamoxifen    and related compounds) and androgens, flutamide, leuprorelin,    goserelin, cyprotrone or octreotide-   g) drugs which target signal transduction in tumour celis including    antibody derivatives such as herceptin;-   h) alkylating drugs such as platinum drugs (cis-platin,    carbonplatin, oxaliplatin, paraplatin) or nitrosoureas;-   i) drugs potentially affecting metastasis of tumours such as matrix    metalloproteinase inhibitors;-   j) gene therapy and antisense agents;-   k) antibody therapeutics; and-   l) other bioactive compounds of marine origin, notably the    ecteinascidins such as ET-743.

In one aspect, the present invention relates to compounds of theformula:

wherein:X is independently —CR₂—, —O—, —S—, or —NR—, in which R is independentlyH or an organic group selected from an alkyl group, an alkenyl group, anaryl group, an aralkyl group, and their substituted derivativessubstituted with one or more of a heterocyclic group, an alkoxy group,an hydroxy group, a mercapto group, an optionally protected amino group,a guanidino group, or a halogen group;X₂ is independently CR, O (and R₂ is absent), S (and R₂ is absent), orN, in which R is independently H or an organic group selected from analkyl group, an alkenyl group, an aryl group, an aralkyl group, andtheir substituted derivatives substituted with one or more of aheterocyclic group, an alkoxy group, an hydroxy group, a mercapto group,an optionally protected amino group, a guanidino group, or a halogengroup;Y is —(COR′)_(n)CO—, where n is 0 or 1 and R′ is an organic groupselected from an alkyl group, an alkenyl group, an aryl group, anaralkyl group, and their substituted derivatives substituted with one ormore of a heterocyclic group, an alkoxy group, an hydroxy group, amercapto group, an optionally protected amino group, a guanidino group,or a halogen group;X₁ is O or S;R₁, R₂ and R₄ are each independently H or an organic group selected froman amido group RCONH— or an acyl group RCO— where R is as defined, analkyl group, an alkenyl group, an aryl group, an aralkyl group, andsubstituted derivatives substituted with one or more of a heterocyclicgroup, an alkoxy group, an hydroxy group, a mercapto group, anoptionally protected amino group, a guanidino group, or a halogen group,and R₁ or R₂ when X₂ is N, and R₄, can further be —SO₂R, where R is asdefined;or R₁ and R₂ with X₂ may form an optionally N-substituted proline, theN-substituted proline aa8 being of formula

where R₃ is independently H or an organic group selected from a groupRSO₂— or an acyl group RCO—, where R is as defied, or R₃ is an alkylgroup, an alkenyl group, an aryl group, an aralkyl group, andsubstituted derivatives substituted with one or more of a carbonylgroup, an hydroxy group, a mercapto group, an optionally protected aminogroup, a guanidino group, or a halogen group;or R₁ and R₂ with X₂ may form a cycloalkyl, aryl or heterocyclic group,optionally substituted with one or more groups R₃;or R₁, R₂, X₂, R₄ and the nitrogen bearing R₄ may form anoxadiazaspiroalkane N-substituted with R₅, where R₅ is independently Hor an organic group selected from a group RSO₂— or an acyl group RCOwhere R is as defined, an alkyl group, an aryl group, an aralkyl group,and substituted derivatives substituted with one or more of a carbonylgroup, an alkoxy group, an hydroxy group, a mercapto group, an aminogroup, a guanidino group, or a halogen group;or aa8 is replaced by an organic group selected from a group RSO₂— or anacyl group RCO where R is as defined, an alkyl group, an aryl group, anaralkyl group, and substituted derivatives substituted with one or moreof a carbonyl group, an alkoxy group, an hydroxy group, a mercaptogroup, an amino group, a guanidino group, or a halogen group;and pharmaceutically acceptable salts thereof.

Preferred compounds include those wherein X is —NR—, in which R is asdefined. More preferably, X is —NH— or —NMe—, and most preferably X is—NH—.

Further preferred compounds include those wherein X is —O—.

The group Y is preferably —COR′CO—, where R′ is an alkyl group,especially where R′ is —CHCH₃—.

Further preferred compounds include those wherein Y is —CO—.

In view of these preferences, a preferred class of compounds is thatwherein X is —NH— or —O— and Y is —COCHCH₃CO— or —CO—.

Preferably R₄ is methyl.

Preferably X₁ is ═O.

Preferably X₂R₁ is an optionally substituted aralkyloxy group, such as abenzyloxy group.

Other preferred compounds include those wherein X₂R₁ is an optionallysubstituted amino group, more preferably those wherein X₂R₁ is a group—NHR₁, where R₁ is an optionally substituted alkyl group, alkenyl group,aryl group, or aralkyl group, especially an alkyl group or an arylgroup, such as a phenyl group or a butyl group.

Further preferred compounds comprise those wherein X₂R₁ is an optionallysubstituted alkyl group, especially where X₂R₁ is a propyl group,isopropyl group, pentyl group or biotin group.

A group of preferred compounds is those wherein —C(═X₂)R₁R₂ form anoptionally substituted amino acid acyl group. Suitably the optionallysubstituted amino acid acyl group is optionally substituted proline oroptionally substituted glycine or optionally substituted valine, andmore especially the optionally substituted proline is optionallysubstituted norvaline-proline, optionally substituted alanine-proline,Boc-proline, optionally substituted alkyl-proline, or the optionallysubstituted glycine is heterocyclic-substituted glycine, or theoptionally substituted valine is valine, Boc-valine, or alkyl-valine.Preferably the optionally substituted proline is norvaline-proline,Z-norvaline-proline, alanine-proline, Z-alanine-proline,Boc-alanine-proline, isobutyrylproline or optionally protectedD-lactylproline, or the heterocyclic-substituted glycine iscoumarinyl-glycine, or the optionally substituted valine is valine,Boc-valine, or isobutyrylvaline.

A further group of preferred compounds includes those wherein X₁ is Sand X₂R₁ is a group —NHR₁, where R₁ is an optionally substituted alkylgroup, alkenyl group, aryl group, or aralkyl group. R₁ is preferably analkyl group or an aryl group, more preferably a phenyl group or a butylgroup.

R₁ and R₂ with X₂ can form a heterocyclic group, optionally substitutedwith one or more groups R₃. For example, the heterocyclic group can becoumarin.

Preferred compounds include those wherein aa8 is replaced by an organicgroup RSO₂—, where R is as defined, such as methyl.

R₁, R₂, X₂, R₄ and the nitrogen bearing R₄ can form anoxadiazaspiroalkane N-substituted with R₅, where R₅ is H. TheN-substituted oxadiazaspiroalkane is preferably6-oxa-1,7-diazaspiro[4,4]nonane.

Examples of compounds according to this invention include:

-   3-[Aip]-Z-didemnin A,-   8-[Phenylurea]-didemnin A,-   8-[Butylurea]-didemnin A,-   3-[val]-8-[isobutyryl]-aplidine,-   9-[norvaline]-aplidine,-   3-[Hiv]-9-[Isobutyryl]-aplidine,-   3-[Val]-9-[Isobutyryl)]-aplidine,-   3-[hiv]-8-[isobutyryl]-didemnin A,-   3-[Hiv]-9-[Ala]-aplidine,-   3-[Hiv]-9-[Nva]-aplidine,-   8-[Phenylthiourea]-didemnin A,-   8-[Coumarin]-didemnin A,-   8-[Butylthiourea]-didemnin A,-   3-[Hiv]-9-[D-Lac]-aplidine,-   8-[Methylsulphonyl]-didemnin A,-   3-[val]-Z-didemnin A,-   3-[Hiv]-8-[Val]-didemnin A,-   3-[Hiv]-8-[butyryl]-aplidine,-   3-[val]-didemnin A,-   3-[Hiv]-didemnin A,-   Z-Didemnin A,-   9-[Z-Nva]-aplidine,-   3-[Hiv]-9-[Z-ala]-aplidine,-   8-[Gly]-9-[Coumarin]-didemnin A,-   8-[Biotin]-didemnin A,-   3-[Hiv]-7,8-[Spiro]-9-[Boc]-aplidine,-   3-[Hiv]-Z-didemnin A,-   3-[Hiv]-9-[Z-Nva]-aplidine,-   7,8-[Spiro]-9-[pyr]-aplidine,-   3-[Hiv]-9-[lac(OTBDMS)]-aplidine,-   3-[Hiv]-9-[Boc-Ala]-aplidine,-   7,8-[Spiro]-9-[Boc]-aplidine,-   3-[Hiv]-8-[Boc-Val]-aplidine,-   8-[Val]-9-[Isobutyryl]-didemnin A,-   3-[Hiv]-8-[hexanoyl]-didemnin A,-   3-[Val]-9-[Lac(OTBDMS)]-aplidine,-   3-[Aip]-didemnin A,-   3-[Hiv]-9-[D-Lac(OTBDMS)]-aplidine,-   7,8-[Spiro]-9-[Isobutyryl]-aplidine,-   3-[Hiv]-7,8-[Spiro]-9-[Pyr]-aplidine,-   3-[Hiv]-7,8-[Spiro]-9-[Isobutyryl]-aplidine,-   3-[Hiv]-7,8-[Spiro]-9-[Acryloyl]-aplidine, or-   [Aip]³-aplidine.

In a related aspect, the present invention is directed to compoundshaving the following formulae:

and related structures.

In one particularly preferred embodiment, the present invention providesa synthetic route to the formation of aa3=[Hiv]³ or [Val]³ or [Aip]³, asa part of a series of exceedingly potent and rare antitumor agents whichscheduled slated for clinical trials when adequate quantities becomeavailable, and its simplest isomers, where amino acid residues arepermuted. This process is enantio- and stereocontrolled and fast, takingadvantages of the standard methods of solution-phase syntheticmethodology

The preferred embodiment of the present invention is represented informula I, wherein aa3 are independently α-amino acids of L or Dconfiguration. If applies X is independently C, O, S, or NR; where R isindependently H or an organic group selected from the group consistingof an alkyl group, an aryl group, an aralkyl group, and theirsubstituted derivatives with an hydroxy group, a mercapto group, anamino group, a guanidino group, a halogeno group. Where R mostpreferably have from 1 to about 12 carbon atoms, more preferably 1 toabout 8 carbon atoms, still more preferably 1 to about 6 carbon atoms,and most preferably 1, 2, 3 or 4 carbon atoms. Methyl, ethyl and propylincluding isopropyl are particularly preferred alkyl groups in thecompounds of the present invention. Where aa3 most preferably isα-(α′-hydroxyisovaleryl)propionyl (Hip), (X═O, Y═—COCHCH₃CO—)-serie A,or α-(α′-aminoisovaleryl)propionyl (Aip) (X═NH, Y═—COCHCH₃CO—)-serie N,or valine (X═NH, Y═—CO—)-serie V, or α-hydroxyisovaleryl (X═—O—,Y═—CO—)-serie H, or N-methylvaline (X═NMe, Y═—CO—)-serie M. Wherein aa8are independently α-amino acids of L or D configuration, if applies;wherein X₂ is independently C, O, S, or N an organic group selected fromthe group consisting of an alkenyl, an alkyl group, an aryl group, anaralkyl group, and their substituted derivatives with an hydroxy group,a mercapto independently H or an organic group selected from the groupconsisting of an alkyl group, an aryl group, an aralkyl group, and theirsubstituted derivatives with an hydroxy group, a mercapto group, anamino group, a guanidino group, a halogeno group, wherein R₁, R₂, R₃ andR₄ are each independently H or an organic group selected from the groupconsisting of an alkyl group, an aryl group, an aralkyl group, and theirsubstituted derivatives with an hydroxy group, a mercapto group, anamino group, a guanidino group, a halogen group. Aa8 also can be aproline residue as in formula II. Where R₃ is independently H or anorganic group selected from the group consisting of an alkyl group, anaryl group, an aralkyl group, and their substituted derivatives with anhydroxy group, a mercapto group, an amino group, a guanidino group, ahalogen group.

Where R₃ most preferably can be pyruvic acid, aralkykoxycarbonyl groupor aminoacid or peptides. Alkyl groups preferably have from 1 to about12 carbon atoms, more preferably 1 to about 8 carbon atoms, still morepreferably 1 to about 6 carbon atoms, and most preferably 1, 2, 3 or 4carbon atoms. Methyl, ethyl and propyl including isopropyl areparticularly preferred alkyl groups in the compounds of the presentinvention. As used herein, the term alkyl, unless otherwise modified,refers to both cyclic and noncyclic groups, although cyclic groups willcomprise at least three carbon ring members. Preferred aminoacids areprotected or non protected D or L glycine, valine, leucine, isoleucine,phenylalanine, tyrosine, tryptophan, methionine, cysteine, aspartate,asparagine, glutamic acid, glutamine, lysine, arginine, proline, serine,threonine, histidine and hydroxyproline. Preferred peptides can beformed with the above mentioned aminoacids.

Besides, aa8 and R₄ can be linked through derivatives of a6-oxo-1,7-diazaspiro[4,4]-nonane structure:

where R₅ most preferably can be pyruvic acid, aralkykoxycarbonyl groupor aminoacid or peptides. Alkyl groups preferably have from 1 to about12 carbon atoms, more preferably 1 to about 8 carbon atoms, still morepreferably 1 to about 6 carbon atoms, and most preferably 1, 2, 3 or 4carbon atoms. Methyl, ethyl and propyl including isopropyl areparticularly preferred alkyl groups in the compounds of the presentinvention. As used herein, the term alkyl, unless otherwise modified,refers to both cyclic and noncyclic groups, although cyclic groups willcomprise at least three carbon ring members. Preferred aminoacids areprotected or non protected D or L glycine, valine, leucine, isoleucine,phenylalanine, tyrosine, tryptophan, methionine, cysteine, aspartate,asparagine, glutamic acid, glutamine, lysine, arginine, proline, serine,threonine, histidine and hydroxyproline. Preferred peptides can beformed with the above mentioned aminoacids.

As used herein, the term “organic group” means a hydrocarbon group thatis classified as an aliphatic group, cyclic group, or combination ofaliphatic and cyclic groups (e.g., aralkyl groups). In the context ofthe present invention, the term “aliphatic group” means a saturated orunsaturated linear or branched hydrocarbon. This term is used toencompass alkyl, alkenyl, and alkynyl groups, for example. The term“alkyl group” means a saturated linear or branched hydrocarbon groupincluding, for example, methyl, ethyl, isopropyl, isobutyl, t-butyl,heptyl, docelyl, octadecyl, amyl, 2-ethylhexyl, 2-methylbutyl,5-methylhexyl, and the like. The term “alkenyl group” means anunsaturated, linear or branched hydrocarbon group with one or morecarbon-carbon double bonds, such as a vinyl group. The term “alkynylgroup” means an unsaturated, linear or branched hydrocarbon group withone or more carbon-carbon triple bonds. The term “cyclic group” means aclosed ring hydrocarbon group that is classified as an alicyclic group,aromatic group, or heterocyclic group. The term “alicyclic group” meansa cyclic hydrocarbon group having properties resembling those ofaliphatic groups. The term “aromatic group” or “aryl group” means amono- or polycyclic aromatic hydrocarbon group. The term “heterocycyclicgroup” means a closed ring hydrocarbon in which one or more of the atomsin the ring is an element other than carbon (e.g., nitrogen, oxygen,sulfur, etc.).

Preferred alkoxy groups in the compounds of the present inventioninclude groups having one or more oxygen linkages and from 1 to about 12carbon atoms, more preferably from 1 to about 8 carbon atoms, and stillmore preferably 1 to about 6 carbon atoms, and most preferably 1, 2, 3or 4 carbon atoms.

Suitable heteroaromatic groups in the compounds of the present inventioncontain one, two or three heteroatoms selected from N, O or S atoms andinclude, e.g., coumarinyl including 8-coumarinyl, quinolinyl including8-quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl,thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl and benzothiazol.Suitable heteroalicyclic groups in the compounds of the presentinvention contain one, two or three heteroatoms selected from N, O or Satoms and include, e.g., tetrahydrofuranyl, tetrahydropyranyl,piperidinyl, morpholino and pyrrolindinyl groups.

Suitable carbocyclic aryl groups in the compounds of the presentinvention include single and multiple ring compounds, including multiplering compounds that contain separate and/or fused aryl groups. Typicalcarbocyclic aryl groups contain 1 to 3 separate or fused rings and from6 to about 18 carbon ring atoms. Specifically preferred carbocyclicarykl groups include phenyl including substituted phenyl, such as2-substituted phenyl, 3-substituted phenyl, 2,3-substituted phenyl,2,5-substituted phenyl, 2,3,5-substituted and 2,4,5-substituted phenyl,including where one or more of the phenyl substituents is anelectron-withdrawing group such as halogen, cyano, nitro, alkanoyl,sulfinyl, sulfonyl and the like; naphthyl including 1-naphthyl and2-naphthyl; biphenyl; phenanthryl; and anthracyl.

Optionally protected amino groups can be protected using groups knownfor this purpose. Suitable protecting groups for amines includecarbamates, amides, and other protecting groups, such as alkyl,arylalkyl, sulpho- or halo-arylalkyl, haloalkyl, alkylsilylalkyl,arylalkyl, cycloalkylalkyl, alkylarylalkyl, heterocyclylalkyl,nitroarylalkyl, acylaminoalkyl, nitroaryldithioarylalkyl,dicycloalkylcarboxamidoalkyl, cycloalkyl, alkenyl, arylalkenyl,nitroarylalkenyl, heterocyclylalkenyl, heterocyclyl,hydroxyheterocyclyl, alkyldithio, alkoxy- or halo- or alkylsulphinylarylalkyl, heterocyclylacyl, and other carbamates, and alkanoyl,haloalkanoyl, arylalkanoyl, alkenoyl, heterocyclylacyl, aroyl,arylaroyl, haloaroyl, nitroaroyl, and other amides, as well as alkyl,alkenyl, alkylsilylalkoxyalkyl, alkoxyalkyl, cyanoalkyl, heterocyclyl,alkoxyarylalkyl, cycloaloyl, nitroaryl, arylalkyl, alkoxy- orhydroxyarylalkyl, and many other groups. Such groups may optionally besubstituted with the previously mentioned substituent groups.

References herein to substituted groups in the compounds of the presentinvention refer to the specified moiety that may be substituted at oneor more available positions by one or more suitable groups, e.g.,halogen such as fluoro, chloro, bromo and iodide; cyano; hydroxyl;nitro; azido; alkanoyl such as a C₁₋₆ alkanoyl group such as acyl andthe like; carboxamido; alkyl groups including those groups having 1 toabout °2 carbon atoms or from 1 to about 6 carbon atoms and morepreferably 1-3 carbon atoms; alkenyl and alkynyl groups including groupshaving one or more unsaturated linkages and from 2 to about 12 carbon orfrom 2 to about 6 carbon atoms; alkoxy groups having those having one ormore oxygen linkages and from 1 to about 12 carbon atoms or 1 to about 6carbon atoms; aryloxy such as phenoxy; alkylthio groups including thosemoieties having one or more thioether linkages and from 1 to about 12carbon atoms or from 1 to about 6 carbo atoms; alkylsulfinyl groupsincluding those moieties having one or more sulfinyl linkages and from 1to about 12 carbon atoms or from 1 to about 6 carbon atoms;alkylsulfinyl groups including those moieties having one or moresulfonyl linkages and from 1 to about 12 carbon atoms or from 1 to about6 carbon atoms; aminoalkyl groups such as groups having one or more Natoms and from 1 to about 12 carbon atoms or from 1 to about 6 carbonatoms; carbocyclic aryl having 6 or more carbons, particularly phenyl(e.g., R being a substituted or unsubstituted biphenyl moiety); andaralkyl such as benzyl.

As is well understood in this technical area, a large degree ofsubstitution is not only tolerated, but is often advisable. Substitutionis anticipated on the compounds of the present invention. As a means ofsimplifying the discussion and recitation of certain terminology usedthroughout this application, the terms “group” and “moiety” are used todifferentiate between chemical species that allow for substitution orthat may be substituted and those that do not allow or may not be sosubstituted. Thus, when the term “group” is used to describe a chemicalsubstituent, the described chemical material includes the unsubstitutedgroup and that group with O, N, or S atoms, for example, in the chain aswell as carbonyl group or other conventional substitution. Where theterm “moiety” is used to describe a chemical compound or substituent,only a unsubstituted chemical material is intended to be included. Forexample, the phrase “alkyl group” is intended to include not only pureopen chain saturated hydrocarbon alkyl substituents, such as methyl,ethyl, propyl, isobutyl, and the like, but also alkyl substituentsbearing further substituents known in the art, such as hydroxy, alkoxy,amino, carboxyl, carboxamido, halogen atoms, cyano, nitro,alkylsulfonyl, etc. Thus, “alkyl group” includes ether groups,haloalkyls, alcohols, thiols, carboxyl, amines, hydroxyalkyls,sulfoalkyls, etc. On the other hand, the phrase “alkyl moiety” islimited to the inclusion of only pure open chain saturated hydrocarbonalkyl substituents, such as methyl, ethyl, propyl, isobutyl, and thelike.

In a further aspects of this invention, there are provided syntheticmethods.

A method is provided of making a didemnin fragment having the structure

the method comprising coupling Boc-D-allo-Ileu-OH with the lithiumenolate of benzyl acetate.

The carbonyl group of a didemnin fragment of formula:

can be reduced to yield a didemnin fragment having the structure

The hydroxy group of a compound of formula:

can be protected to yield a didemnin fragment having the structure

Further deprotection of the benzyl ester group yields a didemninfragment having the structure

A further method of this invention for making a didemnin fragmentcomprises coupling a first reactant having the structure

and a second reactant having the structure

to yield a didemnin fragment having the structure

wherein X is selected from the group consisting of —O— and —NH—; where Ris an amine protecting group; and where R is a hydroxy protecting group.Suitably X is —O— and R is tert-butyldimethylsilyl; or X is —NH— and Ris Boc.

Another method of this invention for making a didemnin fragmentcomprises coupling a first reactant having the structure

and a second reactant having the structure

to yield a didemnin fragment having the structure

wherein X is selected from the group consisting of —O—, —NMe, and —NH—;where R is an amine protecting group; and where R is H. Suitably X is—O— and R is H.; or X is —NH— and R is Boc; or X is —NMe— and R is Boc.

A method of this invention comprises hydrolyzing the didemnin fragmentof formula:

to yield a didemnin fragment having the structure

wherein X is selected from the group consisting of —OH, and —NH₂

Another method involves hydrolyzing the didemnin fragment

to yield a didemnin fragment having the structure

wherein X is selected from the group consisting of —NH₂ and —NHMe.

A further method is provided of making a didemnin fragment, the methodcomprising coupling a first reactant having the structure

and a second reactant having the structure

to yield a didemnin fragment having the structure

wherein X is selected from the group consisting of —O—, —NMe, and —NH—;where Y is —(COCHCH₃)_(n)CO—; where n is 0 or 1.

A method comprises comprising hydrolyzing the didemnin fragment

to yield a didemnin fragment having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or 1.

Another method of making a didemnin fragment is provided by thisinvention, the method comprising coupling a first reactant having thestructure

and a second reactant having the structure

to yield a didemnin fragment having the structure

A method of this invention involves deprotection of the benzyl estergroup of the didemnin fragment of formula

to yield a didemnin fragment having the structure

According to this invention, a method of making a didemnin fragmentcomprises coupling a first reactant having the structure

and a second reactant having the structure

to yield a didemnin fragment having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or 1.

A method comprises deprotection of the didemnin fragment

to yield a didemnin fragment having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or 1.

A further method of this invention for making a didemnin fragmentcomprises the cyclizing the fragment of formula:

to yield a didemnin analog having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or 1.

A method involves hydrolyzing the didemnin analog

to yield a didemnin analog having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or 1.

A method is further provided of making a didemnin analog, the methodcomprising coupling a first reactant having the structure

and a second reactant having the structure

to yield a didemnin analog having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or 1.

Another method comprises deprotection the didemnin fragment of formula:

to yield a didemnin fragment having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or 1.

A method of making a didemnin fragment comprises the coupling of thefragment having the structure

and a second reactant having the structure

to yield a didemnin fragment having the structure

A further method comprises deprotection the didemnin fragment offormula:

to yield a didemnin fragment having the structure

A method of this invention for making a didemnin analog comprises thecoupling of the didemnin analog of formula:

with the fragment

to yield the didemnin analog having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or 1.

A method of making a didemnin analog comprises the coupling of thedidemnin analog having the structure

and the fragment having the structure

to yield the didemnin analog having the structure

wherein X is selected from the group consisting of —O—, and —NH—, and Ris i-Propyl; wherein X is —O— and R is n-Propyl, and R is n-Pentyl

A method of this invention for making a didemnin analog comprises thecoupling of the didemnin analog having the structure

and the fragment having the structure

to yield the didemnin analog having the structure

A method of this invention involves deprotection the didemnin analog

to yield a didemnin fragment having the structure

A method of making a didemnin analog is provided comprising the couplingof the didemnin analog having the structure

and the fragment having the structure

to yield the didemnin analog having the structure

A method further comprises deprotection the didemnin analog

to yield a didemnin analog having the structure

A method is provided of making a didemnin analog comprising the couplingof the didemnin analog

and isobutyryl chloride to yield the didemnin analog having thestructure

A method of making a didemnin analog is provided comprising the couplingof the didemnin analog having the structure

the fragment having the structure

to yield the didemnin analog having the structure

wherein R is Boc, isobutyryl, pyruvyl, or acryloyl.

A method of making a didemnin analog comprises the coupling of thedidemnin analog having the structure

the fragment having the structure

to yield the didemnin analog having the structure

wherein R is SO₂Me, or Z-Nva.

A method is provided by this invention comprising deprotection thedidemnin analog

to yield a didemnin analog having the structure

wherein R₂ is Nva.

A method of making a didemnin analog is part of this invention,comprising the coupling of the didemnin analog having the structure

and the fragment having the structure

to yield the didemnin analog having the structure

wherein R is Boc, isobutyryl, or pyruvyl.

A method of making a didemnin analog comprises the coupling of thedidemnin analog having the structure

and the fragment having the structure

to yield the didemnin analog having the structure

A method of making a didemnin analog is provided comprising the couplingof the didemnin analog having the structure

and the fragment having the structure

to yield the didemnin analog having the structure

A method of making a didemnin analog comprising the coupling of thedidemnin analog having the structure

and the fragment having the structure

to yield the didemnin analog having the structure

A method of making a didemnin analog is provided comprising the couplingof the didemnin analog having the structure

and methylsulphonyl chloride, to yield the didemnin analog having thestructure

A method of making a didemnin analog comprises the coupling of thedidemnin analog having the structure

and the fragment having the structureX═C═N—Rto yield the didemnin analog having the structure

wherein X is O, and S; wherein R is butyl, and phenyl.

It will be appreciated that these methods are all illustrative of thepresent invention and can be modified as desired. In particular,different protecting groups can be adopted for protection of aminogroups or hydroxy groups. Different reagents can be employed tointroduce intended groups. The substituents may be varied as desired,with particular regard to the general formula for the compounds of thisinvention, and the examples of preferred meanings. The modified methodsare part of this invention.

To the extent that it may be necessary to ensure that this descriptionincludes all of the disclosure in our priority applications, and toensure entitlement to the full extent to the priority dates, we herebyincorporate by reference the content of our GB 0016148.9 and GB0103750.6.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The compounds of the present invention can be prepared synthetically.The methods described here for the synthesis of aplidine and derivativescan also be used for the synthesis of a broad range of didemnins.

The structures of some of the compounds are shown in FIG. 1:

Aa3-aplidine derivatives are synthetic cyclic depsipeptides similar instructure to aplidine (compound II) (aa3=Hip, also known asdehydrodidemnin B) which is a natural didemnin isolated from theascidian Aplidium albicans (FIG. 1). The molecules prepared differs bythe presence of hydroxyisovaleric acid (Hiv) (compound I), or valine(compound V) or methyl valine (compound VI) orα-(α′-aminoisovaleryl)propionyl (Aip) (compound VII) instead of thehydroxyisovalerylpropionic (Hip) (II) unit which is present in all othernaturally occurring didemnin congeners. The similarity between these twostructures has also been found recently between didemnin B (compoundIII), the most well-known member of this class of depsipeptides, and anew isolated cyclic depsipeptide from an unidentified Brazilian ascidianof the family Didemnidae: Tamandarine A (compound IV). Compounds I, V,VI and VII are non natural didemnin derivatives.

The structural homology between III and IV is also reflected in theirrespective biological activity. Comparing both compounds, IV retainssimilar levels of in vitro antitumor activity in clonogenic assays aswell as protein biosynthesis inhibition properties, and it has beenshown to be somewhat more active in vitro than III against pancreaticcarcinoma. However, compound IV does not show any tumor type specificitywhatsoever in NCI 60 cell panel. Didemnin B proved to be toxic at dosesnear those required for therapeutic applications and it is likely thatIV is a broad spectrum toxin rather than a selective agent.

[Hiv]³-aplidine (I) otherwise exhibits the same benefits found inaplidine (II) with respect to didemnin B (III), in that is more specificagainst solid tumors like colon, chondrosarcoma and osteosarcoma. in theMTS assay. [Val]³-aplidine (V) and [MeVal]³-aplidine (VI) are otherwisenew compounds which exhibit a high level of in vitro antitumor activity.Finally, compounds V, VI and VII are likely to result, with respect tothe parent aplidine, in an increase in hydrogen bonding at the activesite, and thus, provide more active compounds. In addition the presenceof the amide bond replacing the ester bond may improve the stability ofthe cyclodepsipeptide core.

We report here the first total synthesis of the different series ofaplidine derivatives. By way of example the retrosynthetic analysis isshown in FIG. 2.

The key steps include an efficient macrocyclization of linear precursors6, and a practical stereoselective synthesis of Ist-aa3-Leu-Pro unit(A1), and the right fragment D1. Final coupling of the macrocycle 4 withdifferent side chains affords aa3-aa8-aplidine and derivatives. Therobustness of this synthetic methodology has been proved successfully indeveloping a practical synthesis of aplidine II (aa3=Hip).

The formation of the macrocyclic core is the essential key step in allseries. Successful cyclization at all of the four possible amide bondshas been achieved in previous syntheses of the didemnins. However, inthe present work, the bond linking N(Me)-O(Me)-Tyr and Pro was selectedas the point for macrocyclization based on previous work developedduring the total synthesis of aplidine II (G. Jou, I. Gonzalez, F.Albericio, P. LLoyd-Williams, E. Giralt., J. Org. Chef 1997, 62, 354-366and patent ES-2102322).

The macrocyclic core of the target molecule is disconnected intoIst-aa3-Leu-Pro tetrapeptide left unit A1 and the dipeptideBoc-Thr-(Z-NMe-OMe-Tyr)-OH D1.

Synthesis of the dipeptide right fragment D1 has been already described.However the synthesis outlined in Scheme 1 allows the preparation ofthis intermediate on a kilogram scale. The acid function ofBoc-Thr(OH)—OH was protected with phenacyl bromide to gave directlyalcohol D3, which was esterified with Z-N(Me)-O(Me)-Tyr-OH using DCC inthe presence of DMAP giving D2. Removal of the phenacyl group with Zn inacetic acid afforded cleanly fragment D1.

The formation of precursor A1 is outlined in Scheme 2 for the differentseries. For the SHPL, SVPL and SMPL series, the first coupling stepbetween Leu-Pro-OBzl (A5) and commercially available acid (B1) gavedirectly alcohol A3 ready for the next reaction with isostatine (C1).For the synthesis of SAPL and SNPL series, the route differs to thatpreviously described in that B1 is a β-ketoester synthesized fromα-(α′-hydroxyiso-valeryl)propionic and α-(α′-aminoisovaleryl)propionicacid respectively. The synthesis of the fragment B2 (SAPL and SNPLseries) is outlined in scheme 3. Hydrogenolisis from B2 to B1 isachieved just before the (one pot) coupling reaction with A5.

The preparation of the entire fragment A1 rely on the availability ofthe isostatine portion which has been prepared from the nonproteinogenic amino protected Boc-D-alloisoleucine (C5) on largequantities. The synthetic route for the preparation of C1 is outlined inScheme 4.

Activation of the carboxylic functionality of Boc-D-allo-Ile withcarbonyldiimidazol followed by condensation with the lithium enolate ofthe benzyl acetate, gave the □-ketoester (C4). Stereoselective reductionwith KBH₄ in methanol iven C3. Protection of the secondary hydroxylgroup as the TBDMS ether (C2) and hydrogenolysis of the resulting benzylester afforded isostatine (C1).

Subsequent steps yielding compounds I, II, V, and VII are depicted inscheme 5.

Coupling of fragments A1 and D1 with HBTU/HOBt afforded the linearprecursor type 7. Hydrogenolysis of Cbz and Benzyl protecting groupsproceeds out cleanly and smoothly with Pd(OH)₂ to give 6.Macrocyclization step using HATU/HOAt afforded intermediate 5 in goodyield (75%). Hydrogen chloride in dioxane was used to cleave Bocprotecting group affording amine 4. This compound was coupled withZ-NMe-D-Leu-OH to give 3, which was subjected to hydrogenolysis withPd—C. The resulting compound 2 was coupled with Pyr-Pro-OH side chainusing DIPCDI to afford the corresponding compounds.

Interestingly, precursors of type 2 which are analogs in all aspectsmentioned earlier, to didemnin A have served as starting building blocksfor the synthesis of some interesting congeners, since the N-terminus, afree secondary amino group, offers a site to attach various acyl groupsto the cyclic depsipeptide.

In an earlier study (Rinehart et al J. Med. Chem., 1996, 39, 2819-2834)acyl derivatives of didemnin A (dA) 3 were have also found active as theparent compounds 2. For the aa3 series we found also activity incompounds 3

Tables 1 and 2 show the IC₅₀ values found in compounds 1. TABLE 1Cytotoxicity of [aa3]-aplidine Congeners IC₅₀ (Molar) Compound/SerieP388 A549 HT29 MEL28 DU145 Aplidine (II)/ 1,80E-10 1,80E-10 4,50E-104,50E-10 SAPL1 [Hiv]³-aplidine 4,74E-10 4,74E-10 4,74E-10 4,74E-10(I)/SHPL1 [Val]³-aplidine 1,13E-10 1,13E-10 1,13E-10 1,13E-10 (V)/SVPL1[Aip]³-aplidine 9,01E-10 9,01E-10 (VII)/SMPL1Methodology: after Berjeron et al, Biochem and Bioph Res. Comm., 1984,121, 3, 848-854.

P388=Murine lymphoma. A549=human lung carcinoma. HT-29=human coloncarcinoma. MEL-28=human melanoma. DU145=human prostate carcinoma TABLE 2IC₅₀ (Molar) values for the Aplidine Family Didemnin B Aplidine [Hiv]³-9LSAPL1 SAPL1 Aplidine Line III II SHPL1 I Solid Tumors Bladder 56372.50E−08 3.59E−08 9.02E−08 Breast MX-1 1.54E−06 1.67E−07 N/A Colon HT-298.07E−08 6.87E−07 1.02E−08 Gastric Hs746t 6.60E−09 2.52E−08 7.16E−08Liver SK-HEP-1 9.21E−08 9.44E−08 2.65E−07 NSCL A549 1.21E−04 2.40E−05N/A Ovary SK-OV-3 1.63E−07 7.20E−08 — Pancreas PANC-1 1.52E−10  1.7E−07— Pharynx FADU 9.79E−08 7.29E−08 3.71E−08 Prostate PC-3 9.00E−08  5.13-07 — Prostate DU-145 — — N/A Prostate LNCAP — — 1.46E−08 Renal786-O 2.90E−07 8.31E−08 — SCL NCl- — — N/A H187 Retinoblastoma Y-79 — —— Melanoma Mel-28 — — 4.86E−07 Fibrosarcoma SW 694 3.28E−06 1.49E−06 N/AChondrosarcoma CHSA — — 3.45E−09 Osteosarcoma OSA-FH — — 5.89E−09Leukemias/ Lymphomas ALL (Promyelocytic HL-60 1.44E−07 7.89E−08 N/Aleukemia) ALL (Acute Molt 3 5.45E−07 5.95E−07 1.77E−08 lymphobalstic)CML (Chronic K562 3.31E−06 5.72E−07 5.21E−07 myelogenous) ALL (B-cell)CCRF- 6.55E−07 4.72E−07 — SB Leukemia Mo-B — — — (Hairy B-cell) LeukemiaARH-77 — 1.78E−07 — (Plasma cell) Lymphoma (T cell) H9 2.13E−07 5.25E−07N/A Lymphoma Hut 78 3.56E−08 4.47E−08 — (Cutaneous T cell) LymphomaMC116 8.84E−09 9.21E−07 3.82E−07 (undifferentiated) Lymphoma RAMOS — — —(Burkitts B cell) Lymphoma U-937 1.87E−07 5.62E−07 — (Histiocytic)Lymphoma (B cell) MoB — — — Lymphoma P3HR1 5.58E−08 5.34E−08 — (Burkittsascites) Methodology: MTT MTT MTS (new)N/A = not active

Compounds prepared herein are shown in Schemes 5 and 6. These compoundsinclude N^(α)-propionyl-[aa]³-dA, N^(α)-butyl-[aa]³-dA,N^(α)-isobutyl-[aa]³-dA, and N^(α)-pentanoyl-[aa]³-dA.

Analogs which have two acyl subunits after the N-terminus of the aa3-dAcore were prepared to examine the structural factors contributing to thespecificity to certain tumors. The diacyl compounds isobutyl-Pro-OH,O-isobutyryl-Lac-Pro-OH, N-Benzyl-Ala-Pro-OH were prepared and condensedwith type 2 compounds by the DIPCDI method to obtain respectively, afterdeprotection and purification, [aa]-³-[isobutyryl]⁹-aplidine,[O-isobutyryl-Lac]⁹-aplidine, [Ala]⁹-aplidine.

Preferred Aplidine derivatives: Compounds from Series: H, V

Spirocompounds were also linked to form active compounds:

The spirocyclic fragments synthesis is outlined in scheme 7. Thesynthesis started from the previously reported compound 8. Ref: a)Seebach, D. et al. J. Am. Chem. Soc. 1983, 105, 5390-5398. b) Genin, M.J. et al. J. Org. Chem. 1993, 58, 2334-2337.

Preferred Aplidine derivatives: Compounds from Serie A

Spiro compounds were also linked as in the previous series to giveactive compounds:

TABLE 3 Cytotoxicity of aplidine derivatives IC₅₀ (Molar) SerieDescription MolW P388 A549 SNPL3 3-[Aip]-Z-didemnin A 1076 9.29E−118PUSAPL1 8-[Phenylurea]-didemnin A 1062 9.42E−11 8BUSAPL18-[Butylurea]-didemnin A 1042 9.59E−11 8ISVPL13-[val]-8-[isobutyryl]-aplidine 956 1.05E−10 9NVSAPL19-[norvaline]-aplidine 1139 4.39E−10 9ISHPL13-[Hiv]-9-[Isobutyryl]-aplidine 1054 4.74E−10 9ISVPL13-[Val]-9-[Isobutyryl]-aplidine 1053 4.75E−10 8ISHPL13-[hiv]-8-[isobutyryl]-didemnin A 957 5.22E−10 9ASHPL13-[Hiv]-9-[Ala]-aplidine 1091 9.16E−10 8PSHPL23-[Hiv]-8-[Boc-Pro]-didemnin A 1084 9.22E−10 9NVSHPL13-[Hiv]-9-[Nva]-aplidine 1083 9.23E−10 8PTSAPL18-[Phenylthiourea]-didemnin A 1078 9.27E−10 8CSAPL18-[Coumarin]-didemnin A 1062 9.41E−10 8BTSAPL18-[Butylthiourea]-didemnin A 1058 9.45E−10 9LSHPL1(L)3-[Hiv]-9-[L-Lac]-aplidine (Tamandarine 1056 9.47E−10 A) 9LSHPL1(D)3-[Hiv]-9-[D-Lac]-aplidine 1056 9.47E−10 9LSVPL1(L)3-[Val]-9-[Lac]-aplidine 1055 9.48E−10 8MSAPL18-[Methylsulphonyl]-didemnin A 1021 9.79E−10 SVPL3 3-[val]-Z-didemnin A1020 9.8E−10  9.8E−10 8PSHPL1 3-[Hiv]-8-[Pro]-didemnin A 984 1.01E−098VSHPL1 3-[Hiv]-8-[Val]-didemnin A 986 1.01E−09 8BSHPL13-[Hiv]-8-[butyryl]-aplidine 957 1.04E−09 SVPL2 3-[val]-didemnin A 8861.1E−09 1.13E−09 SHPL2 3-[Hiv]-didemnin A 886 1.1E−09 1.13E−09 SAPL3Z-Didemnin A 1077 9.3E−09 2.32E−09 9NVSAPL2 9-[Z-Nva]-aplidine 12737.9E−09 3.93E−09 9ZASHPL2 3-[Hiv]-9-[Z-ala]-aplidine 1189 4.21E−09 SAPL2Didemnin A 943 5.30E−09  4.24E−09 8G9CSAPL18-[Gly]-9-[Coumarin]-didemnin A 1172 4.26E−09 8BISAPL18-[Biotin]-didemnin A 1169 4.27E−09 9SBSHPL13-[Hiv]-7,8-[Spiro]-9-[Boc]-aplidine 1096 4.56E−09 SHPL33-[Hiv]-Z-didemnin A 1021 4.9E−09  4.9E−09 9NVSHPL23-[Hiv]-9-[Z-Nva]-aplidine 1217 8.22E−09 9SPSAPL17,8-[Spiro]-9-[pyr]-aplidine 1122 8.55E−09 9LSHPL2(L)3-[Hiv]-9-[lac(OTBDMS)]-aplidine 1170 8.55E−09 9BASHPL23-[Hiv]-9-[Boc-Ala]-aplidine 1155 8.65E−09 9SBSAPL17,8-[Spiro]-9-[Boc]-aplidine 1152 8.68E−09 8VSHPL23-[Hiv]-8-[Boc-Val]-aplidine 1086 9.21E−09 8V9ISHPL18-[Val]-9-[Isobutiryl]-didemnin A 1056 9.46E−09 8HSHPL13-[Hiv]-8-[hexanoyl]-didemnin A 985 1.01E−08 9LSVPL2(L)3-[Val]-9-[Lac(OTBDMS)]-aplidine 1169 1.02E−08 SNPL2 3-[Aip]-didemnin A942 1.06E−08 9LSHPL2(D) 3-[Hiv]-9-[D-Lac(OTBDMS)]-aplidine 1170 8.55E−089SISAPL1 7,8-[Spiro]-9-[Isobutyryl]-aplidine 1122 8.91E−08 9SPSHPL13-[Hiv]-7,8-[Spiro]-9-[Pyr]-aplidine 1066 9.38E−08 9SISHPL13-[Hiv]-7,8-[Spiro]-9-[Isobutyryl]-aplidine 1066 9.38E−08 9SASHPL13-[Hiv]-7,8-[Spiro]-9-[Acriloyl]-aplidine 1064 9.39E−08 SerieDescription HT29 MEL28 DU145 SNPL3 3-[Aip]-Z-didemnin A 9.29E−118PUSAPL1 8-[Phenylurea]-didemnin A 9.42E−11 8BUSAPL18-[Butylurea]-didemnin A 9.59E−11 8ISVPL13-[val]-8-[isobutyryl]-aplidine 1.05E−10 9NVSAPL1 9-[norvaline]-aplidine4.39E−10 9ISHPL1 3-[Hiv]-9-[Isobutyryl]-aplidine 4.74E−10 9ISVPL13-[Val]-9-[Isobutyryl]-aplidine 4.75E−10 8ISHPL13-[hiv]-8-[isobutyryl]-didemnin A 5.22E−10 9ASHPL13-[Hiv]-9-[Ala]-aplidine 9.16E−10 8PSHPL2 3-[Hiv]-8-[Boc-Pro]-didemnin A9.22E−10 9NVSHPL1 3-[Hiv]-9-[Nva]-aplidine 9.23E−10 8PTSAPL18-[Phenylthiourea]-didemnin A 9.27E−10 8CSAPL1 8-[Coumarin]-didemnin A9.41E−10 8BTSAPL1 8-[Butylthiourea]-didemnin A 9.45E−10 9LSHPL1(L)3-[Hiv]-9-[L-Lac]-aplidine 9.47E−10 (Tamandarine A) 9LSHPL1(D)3-[Hiv]-9-[D-Lac]-aplidine 9.47E−10 9LSVPL1(L) 3-[Val]-9-[Lac]-aplidine9.48E−10 8MSAPL1 8-[Methylsulphonyl]-didemnin A 9.79E−10 SVPL33-[val]-Z-didemnin A  9.8E−10  9.8E−10  9.8E−10 8PSHPL13-[Hiv]-8-[Pro]-didemnin A 1.01E−09 8VSHPL1 3-[Hiv]-8-[val]-didemnin A1.01E−09 8BSHPL1 3-[Hiv]-8-[butyryl]-aplidine 1.04E−09 SVPL23-[val]-didemnin A 1.13E−09 1.13E−09 1.13E−09 SHPL2 3-[Hiv]-didemnin A1.13E−09 1.13E−09 SAPL3 Z-Didemnin A 4.64E−09 4.64E−09 9NVSAPL29-[Z-Nva]-aplidine 3.93E−09 3.93E−09 3.93E−09 9ZASHPL23-[Hiv]-9-[Z-ala]-aplidine 4.21E−09 SAPL2 Didemnin A 8.48E−09 1.13E−098G9CSAPL1 8-[Gly]-9-[Coumarin]-didemnin A 4.26E−09 8BISAPL18-[Biotin]-didemnin A 4.27E−09 9SBSHPL13-[Hiv]-7,8-[Spiro]-9-[Boc]-aplidine 4.56E−09 SHPL3 3-[Hiv]-Z-didemnin A 4.9E−09  4.9E−09  4.9E−09 9NVSHPL2 3-[Hiv]-9-[Z-Nva]-aplidine 8.22E−099SPSAPL1 7,8-[Spiro]-9-[pyr]-aplidine  8.9E−09 9LSHPL2(L)3-[Hiv]-9-[lac(OTBDMS)]-aplidine 8.55E−09 9BASHPL23-[Hiv]-9-[Boc-Ala]-aplidine 8.65E−09 9SBSAPL17,8-[Spiro]-9-[Boc]-aplidine 8.68E−09 8VSHPL23-[Hiv]-8-[Boc-Val]-aplidine 9.21E−09 8V9ISHPL18-[Val]-9-[Isobutiryl]-didemnin A 9.46E−09 8HSHPL13-[Hiv]-8-[hexanoyl]-didemnin A 1.01E−08 9LSVPL2(L)3-[Val]-9-[Lac(OTBDMS)]-aplidine 8.55E−09 SNPL2 3-[Aip]-didemnin A1.06E−08 9LSHPL2(D) 3-[Hiv]-9-[D-Lac(OTBDMS)]-aplidine 8.55E−08 9SISAPL17,8-[Spiro]-9-[Isobutyryl]-aplidine 8.91E−08 9SPSHPL13-[Hiv]-7,8-[Spiro]-9-[Pyr]-aplidine 9.38E−08 9SISHPL13-[Hiv]-7,8-[Spiro]-9-[Isobutyryl]- 9.38E−08 aplidine 9SASHPL13-[Hiv]-7,8-[Spiro]-9-[Acriloyl]-aplidine 9.39E−08

-   -   Spiro=[(5R)-1-(substituent at        9)-7-[(1R)-1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane]⁷⁻⁹    -   (1) This compound has been described in the literature: Joc Org.        Chem. 2000, 65, 782-792. Their synthesis was published before        their discovery (l): Org. Lett., 2000, vol 0, No. 0, A-D        Methodology: after Berjeron et al, Biochem and Bioph Res. Comm.,        1984, 121, 3, 848-854        388=Murine lymphoma. A549=human lung carcinoma. HT-29=human        colon carcinoma. MEL-28=human melanoma. DU145=human prostate        carcinoma

LIST OF ABBREVIATIONS

-   Miscellaneous-   AA Amino acid-   Ist Isostatine-   Hip Hydroxyisovalerylpropionic acid-   Hiv Hydroxyisovaleric acid-   Aip Aminoisovalerylpropionic acid-   Lac Lactic acid-   LC Liquid Chromatography-   HPLC High Performance Liquid Chromatography-   TLC Thin Layer Cromatography-   M.p. Melting point-   R_(t) Retention time-   Quant. Quantitative yield-   ESI-MS Electrospray Ionization Mass Spectra-   Protecting Groups-   Bn Benzyl-   Boc tert-Butyloxycarbonyl-   TBDMS tert-Butyldimethylsilyl-   Z Benzyloxycarbonyl-   Pac Phenyl acetic-   Solvents-   THF Tetrahydrofurane-   Hex Hexane-   ACN Acetonitrile-   DCM Dichlorometane-   EtOAc Ethyl acetate-   DMF Dimethylformamide-   MTBE Methyl tertbutyl ether-   Et₂O Diethyl ether-   t-BuOH tert-Butanol-   TFA Trifluoroacetic acid-   MeOH Methanol-   EtOH Ethanol-   IPA Isopropanol-   Reagents-   CDI 1,1′-Carbonyldiimidazole-   HOBt 1-Hydroxybenzotriazole-   HBTU    N-[(1H-Benzotriazol-1-yl)(dimethylamino)methylene]-N-methanaminium    hexafluorophosphate N-oxide-   BOP-Cl Bis(2-oxo-3-oxazolidinyl)phosphinic chloride-   HATU    N-[(dimethylamino)-1H-1,2,3,-triazolo[4,5-b]pyridin-1-ylmethilene]-N-methyl-methanaminium    hexafluorophosphate N-oxide-   HOAt 1-Hydroxy-7-aza-benzotriazole-   DCC Dicyclohexylcarbodiimide-   DIPCDI N,N′-Diisopropylcarbodiimide-   TBAF Tetrabutylammonium fluoride-   AcOH Acetic acid-   p-TsOH p-toluensulphonic acid-   DMAP 4-Dimethylamino pyridine-   NMM N-Methyl morpholine-   DIPEA Diisopropylethylamine-   TEA Triethylamine-   TFA Trifluoroacetic acid    General Procedure

All manipulations were conducted under an inert atmosphere of argon. Allsolvents were reagent grade (used in work-ups) or HPLC grade (used asreaction and or as purification solvent). Anhydrous solvents were useddirectly as supplied by the manufacturer. Tetrahydrofuran was freshlydistilled prior to use to remove stabilizer. All other reagents werecommercial compounds of the highest purity available. Analyticalthin-layer chromatography (TLC) was performed on Merck silica gelaluminium sheets (60, F254) precoated with a fluorescent indicator.Visualization was effected using ultraviolet light (254 nm),phosphomolybdic acid (5% w/v) in 95% ethanol, or vainilline. Proton andcarbon magnetic resonance spectra (¹H, ¹³C-NMR) were recorded on aVarian-300 (300 MHz) Fourier transform spectrometer, and chemical shiftswere expressed in parts per million (ppm) relative to CHCl₃ as aninternal reference (7.26 ppm for ¹H and 77.0 for ¹³C). Multiplicitiesare designated as singlet (s), doublet (d), doublet of doublets (dd),doublet of triplets (dt), triplet (t), quartet (q) multiplet (m), andbroad singlet (bs), and coupling constants (J) were expressed in Hz.Optical rotations (in degrees) were measured with a Jasco P1020polarimeter. Electrospray ionization mass spectra (ESI-MS) were obtainedon a Hewlett Packard Series 1100 MSD. Elemental. Flash columnchromatography was carried out on E. Merck silica gel 60 (240-400 mesh)or RP C18 (40-63 □m) using the solvent systems listed under individualexperiments.

The following procedures describe the synthesis of intermediatesobtained toward aplidine (SAPL), [Aiv]³-aplidine (SNPL), [Hiv]³-aplidine(SHPL), [Val]³-aplidine (SVPL), and [MeVal]³-aplidine (SMPL).

Example 1 Synthesis of Benzyl(4R,5S)-4-(tert-Butoxycarbonylamino)-5-Methyl-3-oxoheptanoate (C4)

To a solution of Boc-D-alloIle-OH (15.26 g, 65.9 mmol) in dry THF (200ml) at 0° C. under argon, was added CDI (16.04 g, 98.96 mmol). After 15min, the mixture was allowed to warm to room temperature, and stirredover a period of 16 h. The resulting solution was cooled to −78° C., andadded via cannula to a well stirred solution of benzyl lithium enolatecooled at −78° C. (625 ml, 0.37 M), [prepared by adding dropwise asolution of benzyl acetate (33.34 ml), in THF (165 ml) to a solution oflithium diisopropylamide (0.36M) in THF/hex 3:1 (642 ml) at −78° C.].The temperature should be kept <−75° C. The reaction mixture was stirredat −78° C. for 60 min. Then, it was allowed to come to −10° C. (30 min),recooled to −78° C. and quenched with saturated aq. ammonium chloride(200 ml), then extracted with DCM (3×500 ml) at room temperature. Thecombined extracts were washed successively with aq sat. NaHCO₃ (500 ml)and brine (200 ml). Drying (Na₂SO₄) followed by removal of solvent gavean oil, which was coated on silica C18 and loaded to the top of aLC-RPC18 [Lichroprep RPC-18 (40-60 microns) column. Elution using agradient ACN-H₂O (60 to 100% ACN)] yielded the product C4 as acolourless oil (16.7 g, 70%). [α]²⁰ _(D)−20.0 (c 1, CHCl₃), TLC: Rf=0.32(Merck, RP-C18, ACN-H₂O 7:3).

¹H NMR (300 MHz, CDCl₃) δ 0.77 (d, 3H), 0.94 (t, 3H), 1.25 (s, 9H), 1.60(m, 1H), 1.90 (m, 2H), 3.58 (s, 2H), 4.47 (dd, 1H), 5.00 (d, 1H), 5.18(s, 2H), 7.35 (bs, 5H).

Example 2 Synthesis of (3S,4R,5S)—N-(tert-Butoxycarbonyl)isostatinebenzyl ester (C3)

C4 (16.7 g, 45.9 mmol) was dissolved in methanol (690 ml) at 0° C.Potassium Borohydride (7.43 g, 137.8 mmol) was added to the stirredsolution and after 30 min the reaction was quenched with aq HCl (0.1 N)to pH 4, and extracted with DCM (300 ml). The extract was washedsuccessively with aq NaHCO₃ (100 ml, sat) and brine (100 ml). Drying(Na₂SO₄) followed by removal of solvent afforded alcohol C3 (15.7 g,93%) as a colourless oil. Rf=0.45 (hex-EtOAc 2:1); [α]_(D)=−9.5 (c 0.76,CHCl₃); Rf=0.45 (EtOAc-Hex 1:2).

¹H NMR (300 MHz, CDCl₃) δ 0.81 (d, 3H), 0.90 (t, 3H), 1.20 (m, 1H), 1.36(m, 1H), 1.40 (s, 9H), 1.90 (m, 1H), 2.55 (dd, 1H), 2.70 (dd, 1H), 3.20(d, 1H), 3.61 (m, 1H), 3.90 (m, 1H), 4.40 (d, 1H), 5.18 (s, 2H), 7.40(bs, 5H).

Example 3 Synthesis of Boc-(3S,4R,5S)-Ist(TBDMS)-OBn (C2)

To a solution of C3 (15.7 g, 42.9 mmol) in dry DMF (65 ml) at 0° C.,imidazol (8.77 g, 128.8 mmol), DMAP (1.57 g, 12.81 mmol), and TBDMS-Cl(19.42 g, 128.8 mmol) were added. The reaction mixture was allowed towarm to room temperature overnight, then it was partitioned between Et₂O(200 ml) and successively with aq HCl (100 ml, 0.1 N), aq NaHCO₃ (100ml, sat) and brine (50 ml). After drying (Na₂SO₄) and solvent removal,the residue was purified by flash LC (silica gel, hex) to yield C2(19.96 g, 97%).

[α]_(D)=10.6 (c 1.01, CHCl₃); Rf=0.73 (EtOAc-Hex 1:2).

¹H NMR (300 MHz, CDCl₃) δ 0.15 (s, 6H), 0.82 (s, 9H), 0.85 (d, 3H), 0.89(t, 3H), 1.18 (m, 1H), 1.35 (m, 1H), 1.41 (s, 9H), 1.77 (m, 1H), 2.45(dd, 1H), 2.60 (dd, 1H), 3.62 (m, 1H), 4.20 (m, 1H), 4.40 (d, 1H), 5.05(d, 1H), 5.15 (d, 1H), 7.40 (bs, 5H).

Example 4 Synthesis of Boc-(3S,4R,5S)-Ist(TBDMS)-OH (C1)

To a solution of C2 (10.48 g, 21.8 mmol) in THF (110 ml), degassed andpurged with argon, was added Pd/C 10% (2.096 g, 20% by weight). Themixture was stirred under H₂ (1 atm) for 16 h, then filtered over a 0.45mm teflon filter and concentrated at reduced pressure to give 7.8 g of acolorless oil. Colorless crystals (6 g, 70%) were obtained aftercrystallization in ACN at −20° C. [α]²⁰ _(D) 1.8 (c 0.594, DCM) [Lit.[α]_(D) 1.74 (c 2.64, CHCl₃). Synthesis, 1991, 2941; Rf=0.45 (MerckHPTLC, RP-C18, ACN-H₂O 8:2).

¹H NMR (300 MHz, CDCl₃) δ 0.18 (s, 6H), 0.82 (s, 9H), 0.85 (d, 3H), 0.89(t, 3H), 1.10-1.20 (m, 2H), 1.42 (s, 9H), 1.80 (m, 1H), 2.50 (m, 2H),3.58 (m, 1H), 4.11 (m, 1H).

Example 5 Synthesis of(2S)-2-tert-Butyl(dimethyl)silyloxy-3-methylbutyric acid (SAPLB4)

To a stirred solution of (S)-2-hydroxy-3-methylbutyric acid (21.12 g,181.9 mmol) in DMF (91 ml) at 0° C. under argon were added Imidazol(27.24 g, 400.11 mmol) and DMAP (6.94 g, 54.56 mmol). After 5 min,tert-Butyldimethylchlorosilane (60.31 g, 400.11 mmol) was added. Themixture was allowed to warm to 23° C. and stirred overnight. The crudereaction mixture was partitioned between Et₂O (250 ml) and aq HCl (250ml, 0.1N). The organic phase was washed successively with aq. NaHCO₃(250 ml, sat), and brine (250 ml), dried (Na₂SO₄), filtered andconcentrated at reduced pressure to afford the bissilylated product as apale yellow oil. A solution of this product in THF (100 ml) was addeddropwise (10 min) to a cooled (0° C.) solution of KOH (30.47 g, 543mmol) in THF/H₂O (543 ml: 181 ml). After 40 min the reaction mixture waspartitioned between H₂O (300 ml) and Et₂O (500 ml). The aqueous phasewas partitioned between cold (0° C.) aq HCl (200 ml, 3N) and EtOAc(5×250 ml). The combined organic extracts were dried (Na₂SO₄) filteredand concentrated under reduced pressure to afford SAPLB4 as a paleyellow oil (38.38 g, 91%).

¹H NMR (300 MHz, CDCl₃) δ 0.02 (m, 6H), 0.90 (m, 15H), 2.08 (m, 1H),4.06 (d, 1H).

Example 6 Synthesis of Benzyl(4S)-4-tert-Butyl(dimethyl)silyloxy-5-Methyl-3-oxohexanoate (SAPLB3)

To a solution of (S)-2-(tertButyldimethylsilyloxy)-3-methylbutyric acid(15.31 g, 65.9 mmol) in dry THF (200 ml) at 0° C. under argon, was addedCDI (16.04 g, 98.96 mmol). After 15 min, the mixture was allowed to warmto room temperature, and stirred over a period of 16 h. The resultingsolution was cooled to −78° C., and added via cannula to a well stirredsolution cooled at −78° C. of benzyl lithium enolate (625 ml, 0.37 M),[prepared by adding dropwise a solution of benzyl acetate (33.34 ml), inTHF (165 ml) to a solution of lithium diisopropylamide (0.36M) inTHF/hex 3:1 (642 ml) at −78° C.]. The temperature should be kept <−75°C. The reaction mixture was stirred at −78° C. for 60 min. Then, it wasallowed to come to −10° C. (30 min), recooled to −78° C. and quenchedwith aq. ammonium chloride (200 ml, sat), then extracted with DCM (3×500ml) at room temperature. The combined extracts were washed successivelywith aq NaHCO₃ (500 ml, sat) and brine (200 ml). Drying (Na₂SO₄)followed by removal of solvent gave an oil, which was coated on silicaC18 and loaded to the top of a LC-RPC18 [Lichroprep RPC-18 (40-60microns), column. Elution using a grad. ACN-H₂O (60 to 100% ACN)]yielded SAPLB3 as a colourless oil (16.1 g, 70%). [α]_(D)−25 (c 0.5,MeOH); Rf=0.32 (Merck, RP-C18, ACN-H₂O 7:3).

¹H NMR (300 MHz, CDCl₃) δ 0.02 (m, 6H), 0.92 (m, 15H), 1.92 (m, 1H),3.63 (s, 2H), 3.80 (s, 2H), 3.38 (d, 1H), 5.17 (d, 1H), 5.20 (d, 1H),7.35 (bs, 5H).

Example 7 Synthesis of Benzyl(4S)-4-tert-Butoxycarbonylamino-5-Methyl-3-oxohexanoate (SNPLB3)

Following the procedure obtained for the synthesis of SAPLB3 fromBoc-Val-OH (10 g, 46.0 mmol), the title compound was obtained afterpurification by flash LC (silica gel, gradient hex-EtOAc 10:1 to 5:1) asan oil (6.9 g, 43%).

¹H NMR (300 MHz, CDCl₃) δ 0.77 (d, J=7, 3H), 0.98 (d, J=7, 3H), 1.44 (s,9H), 2.22 (m, 1H), 3.58 (s, 2H), 4.31 (m, 1H), 5.03 (m, 1H), 5.18 (s,2H), 7.34 (bs, 5H).

ESI-MS Calcd for C₁₉H₂₇NO₅: 349.19. Found (m/z): 372.1 (M+Na)⁺.

Example 8 Synthesis of Benzyl(2RS,4S)-4-tert-butyl(dimethyl)silyloxy-2,5-dimethyl-3-oxohexanoate(SAPLB2)

The ester SAPLB3 (15.12 g, 41.49 mmol) in dry THF (43 ml) was addeddropwise to a solution of lithium diisopropylamine at −78° C. [preparedby adding butyllithium (1.6 M solution in hex; 31.12 ml, 49.79 mmol)dropwise to diisopropylamine (7.26 ml, 51.86 mmol) in dry THF (83 ml)under Ar at −78° C. for 30 min.] The mixture was stirred for 0.5 h andthen, Iodomethane was added (52.11 ml, 829.8 mmol). The mixture wasallowed to warm to 23° C. and then, stirring continues for 24 h.Additional Iodomethane was added (2.67 ml, 42 mmol) and the mixture wasstirred 24 h further or until disappearance of starting material. Themixture was then partitioned between aq. NH₄Cl (50 ml, sat) and EtOAc(2×200 ml). The organic layer was washed successively with aq. NaHCO₃(100 ml, sat), brine (100 ml), dried (Na₂SO₄), filtered and concentratedto give a yellow oil (12 g). Pure product (SAPLB2) was obtained afterpurification by LC-silica gradient hex-Et₂O 100:0 to 100:2 as adiastereomeric mixture of epimers at C2 (10 g, 63%).

¹H NMR (300 MHz, CDCl₃) δ 0.01 (s, 3H), 0.02 (s, 3H), 0.91 (m, 15H),1.30 (d, 3H), 2.01 (m, H), 4.01 (m, 1H), 5.10 (d, 1H), 5.15 (d, 1H),7.34 (bs, 5H).

Example 9 Synthesis of Benzyl(2RS,4S)-4-tert-butoxycarbonylamino-2,5-dimethyl-3-oxohexanoate (SNPLB2)

Following the procedure obtained for the synthesis of SAPLB2, startingfrom SNPLB3 (10 g, 46.0 mmol), the title compound was obtained afterpurification by flash LC (silica gel, gradient hex-EtOAc 10:1 to 5:1) asa diastereomeric mixture (1:1) of epimers at C2 (4.4 g, 62%). Rf=0.4 and0.37 (silica, Hex/EtOAc 3:1).

¹H NMR (300 MHz, CDCl₃) δ 0.72 (m, 3H), 0.86 (m, 3H), 1.37 (m, 3H), 1.44(s, 9H), 2.22 (m, 1H), 3.79 (m, 1H), 4.43 (m, 1H), 5.02 (m, 1H), 5.16(m, 2H), 7.34 (m, 5H).

ESI-MS Calcd for C₂₀H₂₉NO₅: 363.20. Found (m/z): 364.1 (M+H)⁺.

Example 10 Synthesis of Leu-Pro-OBn as Chlorhydrate Salt (A5)

To a flask containing Boc-Leu-Pro-OBn (113.8 g, 272 mmol) a solution ofhydrogen chloride in dioxane (209 ml, 5.3 N) was added and the stirringwas continued for 5 h or until total conversion by TLC (disappearance ofstarting material: Rf=0.47 (hex-EtOAc 2:1, silica). The solution wasconcentrated under reduced pressure and the resulting oil was chasedwith CHCl₃ (3×50 ml), CHCl₃-MTBE (30 ml-50 ml), MTBE (50 ml) and hex (50ml). The residue was dried under vacuum (16 h) to remove residual HCl,to give the title compound as a white solid. A5 (96.4 g, 100%) was useddirectly without further purification in the next step. [α]_(D) ²²−85.21(c=1, CHCl₃).

¹H NMR (300 MHz, CDCl₃) δ 0.92 (d, J=7.1, 3H), 0.96 (d, J=7.1, 3H), 1.55(m, 1H), 1.82-2.14 (m, 5H), 2.26 (m, 1H), 3.42 (m, 1H), 3.90 (m, 1H),4.32 (bs, 1H), 4.64 (m, 1H), 5.01 (d, J=11.5, 1H), 5.16 (d, J=11.5, 1H),7.34 (m, 5H), 8.40 (bs, 3H).

ESI-MS Calcd for C₁₈H₂₆N₂O₃: 318.19. Found (m/z): 319.2 (M+H)⁺.

Example 11 Synthesis of TBDMS-Hip-Leu-Pro-OBn (SAPLA4)

To a flask containing a degassed solution of SAPLB2 (20 g, 52.88 mmol)in THF anh. (158 ml), provided with gas inlet-outlet tubes, was added10% Pd/C (6.0 g, 30% by wt.) under Ar. Then, a stream of hydrogen ispassed through for 8 h or until complete conversion by TLC(disappearance of starting material). The resulting mixture was bubbledwith Ar to displace hydrogen, and filtered under Ar in a sintered glassfunnel through a sort pad of celite, to a cooled flask (−5° C.)containing HOBt (7.17 g, 52.88 mmol) and HBTU (21.0 g, 55.53 mmol).Additional THF (158 ml) was added to wash the celite. To the mixture (at−5° C.) were added NMM (5.8 ml, 52.88 mmol) and after 5 min a cooled(−5° C.) solution containing: A5 (31.96 g, 89.81 mmol), NMM (16 ml, 145mmol) and DMF (120 ml), fresh prepared. The reaction mixture was allowedto warm to rt and stirred for 14 h. The crude reaction was filtered andthe solvent removed under reduced pressure. To the residual solution ofDMF, EtOAc (300 ml) was added and washed successively with aq HCl (200ml, 0.1 N), aq. NaHCO₃ (200 ml, sat.) and rinsed with brine (300 ml).The organic phase was dried (Na₂SO₄) filtered and concentrated. Theresulting material was coated with silica (EtOAc as solvent), andchromatographed on silica gel eluting with a gradient EtOAc:hex 1:5 to1:1 to yield SAPLA4 (26.8 g, 78%) as a thick colourless oil. Thisproduct is a 1:1 mixture of diastereomers. Rf=0.5 (silica, Hex/EtOAc1:1, dark blue/vainillin).

IR (film, DCM) 3295, 3060 and 3040, 2957, 2934, 2880, 2858, 1736, 1634,1528, 1454, 1387, 1252, 1171, 1070 cm⁻¹.

¹H NMR (300 MHz, CDCl₃) δ 0.01 (s, 3H), 0.03 (s, 3H), 0.85-0.97 (m,12H), 0.92 (s, 9H), 0.93 (s, 9H), 1.33 (d, J=7.0, 3H), 1.37 (d, J=7.0,3H), 2.40-2.65 (m, 3H), 1.92-2.28 (m, 4H), 3.64-3.76 m, 1H), 4.69-4.82(m, 1H), 5.05 (d, J=11.8, 1H), 5.20 (d, J=11.8, 2H), 6.73 (d, J=8.9,1H), 6.98 (d, J=9.0, 1H), 7.34 (bs, 5H).

¹³C NMR (75 MHz, CDCl₃) δ−5.24, −4.81, 15.70, 17.43, 17.57, 18.84,21.48, 21.61, 18.05, 23.28, 24.43, 24.55, 24.76, 25.68, 28.87, 31.36,31.77, 41.27, 41.67, 48.68, 48.55, 48.89, 58.71, 66.84, 83.84, 83.29,128.09, 128.47, 135.40, 169.24, 170.67, 170.89, 171.16, 171.20, 209.11,211.62.

m/z (FAB) 611.5 [(M+Na)⁺, 15], 589.5 [(M+H)⁺, 100]; m/z (FABHRMS)589.369 045, C₃₂H₅₂N₂O₆Si requires (M+H)⁺, 589.367 291

Example 12 Synthesis of Boc-Aip-Leu-Pro-OBn (SNPLA4)

To a degassed solution of SNPLB2 (2.3 g, 6.32 mmol) in dry THF (30 ml)was added 10% Pd/C (0.74 g, 16% by wt.) and then hydrogenated atatmospheric pressure for 5 h. 30 min or until complete conversion by TLC(disappearance of starting material). The resulting mixture was filteredthrough a sort pad of celite and additional THF (20 ml) was added towash the celite. To the filtered solution (at −5° C.) were added BOP-Cl(1.77 g, 6.96 mmol) and NMM (765 □l, 6.91 mmol) and after 30 min acooled (−5° C.) solution containing: A5 (3.15 g, 8.85 mmol), NMM (1.88ml, 8.84 mmol) and DMF (14 ml) prepared 10 min before. The reactionmixture was allowed to warm to rt and stirred for 17 h. The crudereaction was filtered and the solid washed with EtOAc (100 ml). Thecombined organic solutions was successively washed with aq KHSO₄ (50 ml,10%), aq. NaHCO₃ (50 ml, sat.) and brine (50 ml). The organic phase wasdried (Na₂SO₄) filtered and concentrated in vacuo, and the resultingmaterial was chromatographed on silica gel eluting with a gradientEtOAc:hex 1:4 to 1:1 to yield SNPLA4 (750 mg, 20%) as a white solid.This product is a mixture of diastereomers. Rf=0.26 and 0.17 (silica,Hex/EtOAc 1:1).

¹H NMR (300 MHz, CDCl₃) δ 0.74-1.04 (m, 12H), 1.31-1.70 (m, 6H), 1.43(s, 9H), 2.01 (m, 3H), 2.22 (m, 2H), 3.60 (m, 2H), 3.77 (m, 1H), 4.40(m, 1H), 4.58 (m, 1H), 4.69 (m, 1H), 5.14 (m, 2H), 6.75 (d, J=8.7, 1H),7.04 (d, J=7.8, 1H), 7.34 (bs, 5H).

¹³C NMR (75 MHz, CDCl₃) δ 16.74, 16.91, 20.22, 21.94, 23.53, 24.80,25.06, 28.48, 29.14, 41.29, 41.41, 46.98, 49.45, 49.64, 51.26, 59.06,63.70, 64.27, 67.08, 79.86, 80.10, 128.32, 128.48, 128.74, 135.74,156.28, 169.15, 169.32, 171.18, 171.91.

ESI-MS Calcd for C₃₁H₄₇N₃O₇; 573.34. Found (m/z): 574.4 [(M+H)]⁺.

Example 13 Synthesis of Hiv-Leu-Pro-OBn (SHPLA4)

To a flask containing A5 (2.06 g, 4.78 mmol) in DCM (5 ml) at 0° C., NMM(506 mg, 5.01 mmol) was added with stirring. After 15 min(2S)-2-hydroxy-3-methylbutanoic acid (hydroxyisovaleric acid) (487 mg,4.78 mmol) and DCC (986 mg, 4.78 mmol) were added in portions. Thereaction mixture was allowed to warm to 23° C. and stirred for 14 h. Thesuspension was diluted with CHCl₃ (25 ml) and partitioned between aq HCl(10 ml, 1N), aq NaHCO₃ (10 ml, sat) and brine (10 ml) dried (Na₂SO₄) andconcentrated under reduced pressure. The residue was purified by flashLC (silica gel, gradient hex-EtOAc 1:1 to 1:3) to give SHPLA4 (1.13 g,80%) as a white solid. Rf=0.46 (hex-EtOAc 1:2).

¹H NMR (300 MHz, CDCl₃) δ 0.81, (d, J=7.0, 3H) 0.92 (m, 6H), 0.97 (d,J=7.0, 3H), 1.42 (m, 1H), 1.63 (m, 2H), 2.00 (m, 3H), 2.19 (m, 2H), 3.60(m, 1H), 3.85 (m, 1H), 3.88 (d, J=4.8, 1H), 4.46 (m, 1H), 4.80 (m, 1H),5.06 (d, J=12.3, 1H), 5.14 (d, J=12.3, 1H), 7.32 (m, 5H), 7.41 (d,J=8.4, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 15.32, 19.08, 21.38, 23.11, 24.41, 24.69,28.77, 31.31, 40.54, 46.81, 48.20, 58.82, 66.69, 75.76, 127.93, 128.14,128.37, 135.28, 171.39, 171.95, 173.94.

Example 14 Synthesis of Boc-Val-Leu-Pro-OBn (SVPLA4)

To a flask containing Boc-Valine-OH (652 mg, 3 mmol) in DCM (6 ml) at 0°C. was added NMM (0.35 ml, 3.15 mmol). After stirring for 15 min, A5(1.065 g, 3 mmol), HOBt (405 mg, 3.0 mmol) and DCC (650 mg, 3.15 mmol)were added in portions. The reaction mixture was allowed to warm to 23°C. and stirred for 14 h. The suspension was diluted with DCM (25 ml) andwashed successively with aq KHSO₄ (2×10 ml, 10%), aq NaHCO₃ (2×10 ml,sat) and brine (10 ml) dried over Na₂SO₄ filtered and concentrated underreduced pressure. The residue was purified by flash LC (silica, gradienthex-EtOAc 2:1 to 1:1) to give SVPLA4 (1.48 g, 93%) as a white solid.Rf=0.57 (hex-EtOAc 1:2).

¹H NMR (300 MHz, CDCl₃) δ 0.83-0.95 (m, 12H), 1.2-1.4 (m, 1H), 1.42 (s,9H), 1-40-1.51 (m, 1H), 1.60-1.75 (m, 1H), 1.82-2.20 (m, 5H), 3.50-3.60(m, 1H), 3.74-3.78 (m, 1H), 3.91 3.96 (m, 1H), 4.52-4.57 (s, 1H),4.75-4.77 (m, 1H), 5.04 (bs, 1H)—, 5.05 (d, J=12.3, 1H), 5.17 (d,J=12.3, 1H), 6.60 (d, J=8.4, 1H), 7.26-7.35 (m, 5H).

¹³C NMR (75 MHz, CDCl₃) δ 7.61, 19.19, 21.66, 23.23, 24.43, 24.78,24.87, 25.55, 28.20, 28.87, 30.91, 33.86, 41.68, 46.73, 48.86, 58.77,59.74, 66.82, 79.66, 128.08, 128.21, 128.46, 135.47, 155.66, 170.83,171.33, 171.60.

ESI-MS Calcd for C₂₈H₄₃N₃O₆: 517.32. Found (m/z): 518.2 [(M+H)]⁺.

Example 15 Synthesis of Boc-Me-Val-Leu-Pro-OBn (SMPLA4)

Following the procedure described for the synthesis of SVPLA4, startingfrom A5 (1.07 mg, 3.00 mmol) and Boc-(Me)Val-OH (694 mg, 3.00 mmol), thetitle compound was obtained as a white solid (1.39 g, 87%) afterpurification by flash LC (silica gel, gradient hex-EtOAc 2:1 to 1:1).Rf=0.51 (Hex-EtOAc 1:2).

¹H NMR (300 MHz, CDCl₃) δ 0.83-0.91 (m, 12H), 1.45 (s, 9H), 1.93-2.03(m, 4H), 2.18-2.22 (m, 2H), 2.76 (s, 3H), 3.40-3.50 (m, 2H) 3.55-3.62(m, 1H), 3.75-3.85 (m, 1H), 4.00-4.10 (m, 1H), 4.50-4.60 (m, 1H),4.70-4.82 (m, 1H), 5.07 (d, J=11.2, 1H), 5.24 (d, J=11.2, 1H), 6.20 (m,0.5H), 6.50 (m, 0.5H), 7.26-7.35 (m, 5H).

¹³C NMR (75 MHz, CDCl₃) δ 18.43, 19.77, 21.53, 23.23, 24.47, 24.77,25.89, 25.30, 28.85, 41.25, 46.67, 48.51, 58.75, 64.05, 66.79, 128.06,128.18, 128.44, 135.49, 170.12, 170.80, 171.660

ESI-MS Calcd for C₂₉H₄₅N₃O₆: 531.33. Found (m/z): 532.3 (M+H)⁺.

Example 16 Synthesis of Hip-Leu-Pro-OBn (SAPLA3)

To a flask containing SAPLA4 (15.86 g 26.97 mmol) a clear colorlesssolution of tetrabutylammonium fluoride 1M in THF (80.9 ml, 80.9 mmol)was added and the mixture was stirred vigorously at r.t. for 15 min (ortotal conversion by TLC). The reaction was quenched by addition of H₂O(4 ml) and silica gel (50 g). The crude material was concentrated andpurified by flash LC (silica gel, grad hex:EtoAc 2:1 to 1:1) to yieldSAPLA3 (12.2 g, 95%) as a white solid (mixture of diastereomers).Rf=0.36 and 0.29 (silica, hex:CHCl₃:IPA; 1:5:1).

IR (film, DCM) ν 3450-3293, 3060 and 3040, 2961, 2946, 2883, 2852, 1746,1632, 1533, 1454, 1357, 1387, 1265, 1173, 1095, 1045, 1018 cm⁻¹.

¹H NMR (500 MHz, CDCl₃) δ 0.71 (d, J=6.8, 3H), 0.81 (d, J=6.6, 3H), 0.88(d, J=6.5, 3H), 0.91 (d, J=6.5, 3H), 0.94 (d, J=6.5, 3H), 0.99 (d,J=7.1, 3H), 1.07 (d, J=6.5, 3H), 1.36 (d, 6.5, 3H), 1.43-1.52 (m, 2H),1.60-1.66 (m, 1H), 1.93-2.10 (m, 3H), 2.12-2.23 (m, 2H), 3.53-3.58 (m,1H), 3.65 (q, J=7.1, 1H), 3.67-3.73 (m, 1H), 3.89 (q, J=7.1, 1H), 3.96(d, J=4.2, 1H), 4.22 (d, J=4.1, 1H), 4.54-4.56 (m, 1H), 4.58-4.62 (m,1), 4.69-4.73 (m, 1H), 5.1 (d, J=12.1, 1H), 5.18 (d, J=12.1, 1H), 6.57(d, J=8.5, 1H), 6.63 (d, J=8.5, 1H), 7.28-7.38 (m, 5H).

¹³C NMR (75 MHz, CDCl₃) δ 14.06, 14.26, 15.85, 16.48, 20.07, 20.53,22.02, 22.25, 25.37, 25.46, 29.45, 29.53, 31.59, 32.09, 41.13, 42.29,49.93, 50.91, 51.02, 59.52, 67.60, 81.02, 128.78, 1.29.2, 169.48,171.58, 172.17, 209.76.

m/z (FAB) 497.4 [(M+Na)⁺, 12], 475.5 [(M+H)⁺, 100]. m/z (FABHRMS)497.263 162, C₂₆H₃₈N₂O₆ requires (M+Na)⁺ 497.262 757. Anal. Calcd forC₂₆H₃₈N₂O₆: C, 65.82; H, 8.02; N, 5.91. Found: C, 65.97; H, 8.18; N,5.76.

Example 17 Synthesis of Aip-Leu-Pro-OBn (SNPLA3)

To a solution of SNPLA4 (750 mg, 1.30 mmol) in dioxane (15 ml, anh), asolution of hydrogen chloride in dioxane (39 ml, 5.3 N) was added andthe mixture was stirred for 5 hours or until total conversion by TLC(disappearance of starting material). The solution was concentratedunder reduced pressure and the resulting oil was chased with CHCl₃ (15ml), MTBE (15 ml) and hex (15 ml). The residue was dried under vacuum toremove residual HCl, to give a foamy solid. SNPLA3 (660 mg, quant.) wasused directly in next step with no further purification.

ESI-MS Calcd for C₂₆H₃₉N₃O₅: 473.29. Found 474.2 (M+H)⁺.

Example 18 Synthesis of Val-Leu-Pro-OBn (SVPLA3)

To a flask containing SVPLA4 (215 mg, 0.41 mmol) a solution of hydrogenchloride in dioxane (1.5 ml, 5.3 N) was added and the mixture wasstirred for 5 hours or until total conversion by TLC. The solution wasconcentrated under reduced pressure and the resulting oil was chasedwith CHCl₃ (5 ml), MTBE (5 ml) and hex (5 ml). The residue was driedunder vacuum to remove residual HCl, to give a foamy solid of SVPLA3(185 mg, quant.) was used directly in next step with no furtherpurification.

¹H NMR (300 MHz, CDCl₃) δ 0.86-0.90 (m, 6H), 1.04 (d, J=6.3, 3H), 1.12(d, J=6.3, 3H), 1.45-1.55 (m, 1H), 1-60-1.80 (m, 2H), 1.82-2.11 (m, 2H),2.11-2.25 (m, 1H), 2.25-2.40 (m, 1H), 3.50-3.70 (m, 1H), 3.80-3.95 (m,2H), 4.52-4.57 (s, 1H), 4.70-4.85 (m, 1H), 5.05 (d, J=12, 1H), 5.20 (d,J=12.3, 1H), 7.27-7.37 (m, 5H), 7.91 (m, 1H), 8.62 (bs, 2H).

¹³C NMR (75 MHz, CDCl₃) δ 18.57, 18.79, 21.83, 23.11, 24.50, 24.67,24.90, 25.34, 28.92, 30.23, 33.25, 40.40, 47.04, 49.46, 49.94, 59.26,60.02, 66.88, 128.16, 128.27, 128.51, 135.48, 167.54, 170.80, 171.94.

Example 19 Synthesis of (Me)Val-Leu-Pro-OBn (SMPLA3)

Following the procedure described for the synthesis of SVPLA3, startingfrom SMPLA4 (940 mg, 1.94 mmol) the title compound (828 mg, quant.) wasobtained as a white solid. This product was used directly in next stepwith no further purification.

¹H NMR (300 MHz, CDCl₃) δ 0.93 (d, J=6.3, 6H), 1.07 (d, J=6.3, 3H), 1.21(d, J=6.3, 3H), 1.47 (m, 1H), 1.73 (m, 2H), 2.00 (m, 3H), 2.23 (m, 1H),2.52 (m, 1H), 2.83 (bs, 3H), 3.56-3.65 (m, 2H), 3.77 (m, 1H), 4.59 (m,1H), 4.66 (m, 1H), 5.07 (d, J=12.3, 1H), 5.19 (d, J=12.3, 1H), 7.27-7.38(m, 5H), 7.90 (m, 1H), 9.11 (m, 0.5H), 9.61 (m, 0.5H).

¹³C NMR (75 MHz, CDCl₃) δ 13.98, 18.37, 19.57, 21.33, 22.52, 23.16,24.74, 28.77, 29.78, 31.54, 32.54, 39.87, 46.75, 50.09, 58.91, 66.85,122.12, 128.06, 128.24, 128.47, 135.43, 166.22, 170.73, 171.54.

□ESI-MS Calcd for C₂₄H₃₈ClN₃O₄: 431.2. Found (m/z): 432.2 (M+H)⁺.

Example 20 Synthesis of Boc-Ist(TBDMS)-Hip-Leu-Pro-OBn (SAPLA2)

To a solution of SAPLA3 (12.2 g, 25.44 mmol) in anh. DCM (75 ml) at −5°C. under Ar, DMAP (0.932 g, 7.6 mmol), C1 (11.89 g, 30.53 mmol) and DCC(6.613 g, 32.05 mmol) were added in portions, while maintaining thetemperature <−5° C. (ice-salt bath). The reaction mixture was stirredfor 14 h at −5° C. and then, filtered and concentrated. The crudematerial was chased with ACN, cooled (−10° C.), filtered andconcentrated again. The resulting material was dissolved in EtOAc (400ml) and washed sequentially with aq. KHSO₄ (2×200 ml, 10%), brine (200ml), aq. NaHCO₃ (200 ml, sat.) and rinsed with brine (200 ml). Theorganic phase was dried (Na₂SO₄), filtered and concentrated at reducedpressure to afford a colourless oil which was chromatographed on silicagel eluting with a gradient of Hex-EtOAc 3:1 to 2:1, to yield SAPLA2(19.35 g, 90%) as a white foam (mixture of diastereomers).

IR (film, DCM) ν 3365-3200, 3069, 3038, 2959, 2930, 2882, 2857, 1746,1688, 1640, 1533, 1456, 1389, 1258, 1171, 1086 cm⁻¹.

¹H NMR (500 MHz, CDCl₃) δ 0.01 (s, 3H), 0.03 (s, 3H), 0.05 (s, 3H), 0.07(s, 3H), 0.77-1.03 (m, 18H), 0.84 (s, 9H), 0.85 (s, 9H), 1.33 (d, J=7.4,3H), 1.32-1.36 (m, 2H), 1.49 (d, J=7.5, 3H), 1.38-1.62 (m, 3H), 1.42 (s,9H), 1.44 (s, 9H), 1.51-1.77 (m, 1H), 1.88-2.37 (m, 3H), 2.17-2.33 (m,2H), 2.47-2.74 (m, 2H), 3.34-3.72 (m, 1H), 3.72-3.82 (m, 1H), 3.99-4.40(m, 1H), 4.03-4.16 (m, 1H), 4.49 (d, J=10.3, 1H), 4.54-4.59 (m, 1H),4.63-4.70 (m, 2H), 4.75 (d, J=4.5, 1H), 4.77-4.81 (m, 1H), 4.95-5.19 (m,2H), 5.22 (d, J=5.2, 1H), 5.32 (d, J=10.5, 1H), 6.38 (d, J=10.9, 1H),6.71 (d, J=7.4, 1H), 6.76 (d, J=8.4, 1H), 8.60 (d, J=9.5, 1H).

¹³C NMR (75 MHz, CDCl₃) δ−5.05, −4.49, 11.83, 12.03, 13.01, 13.51,13.83, 14.08, 16.92, 17.10, 17.85, 19.14, 19.65, 21.57, 22.09, 22.96,23.28, 24.36, 24.60, 24.85, 25.73, 26.97, 27.33, 28.35, 28.46, 28.93,29.09, 29.65, 34.12, 34.16, 40.45, 40.85, 41.18, 42.20, 46.74, 46.16,47.99, 48.34, 48.90, 49.42, 57.62, 58.81, 58.96, 60.46, 66.62, 66.88,68.18, 69.69, 78.98, 79.24, 79.84, 82.95, 128.08-128.49, 135.48 135.61,155.85, 158.27, 157.44, 168.40, 169.07, 170.65, 170.86, 171.42 171.79,203.09 205.97.

m/z (FAB) 846.6 f(M+H)⁺, 151, 746.6 (100); m/z (FABHRMS) 868.516 630,C₄₅H₇₅N₃O₁₀Si requires (M+Na)⁺ 868.511 930.

Example 21 Synthesis of Boc-Ist(TBDMS)-Aip-Leu-Pro-OBn (SNPLA2)

To a flask containing SNPLA3 (chlorhydrate) (660 mg, 1.12 mmol) in DCM(15 ml, anh) at 0° C., NMM (0.19 ml) was added. After 15 min, C1 (632mg, 1.62 mmol), HOBt (266 mg, 1.73 mmol), and DCC (331 mg, 1.60 mmol)were added in portions. The flask was allowed to warm to roomtemperature and stirring was continued overnight. Crude reaction mixturewas partitioned between DCM (50 ml) and aq KHSO₄ (2×20 ml, 10%). Theorganic phase was washed successively with aq. NaHCO₃ (2×20 ml, sat) andbrine (20 ml), dried (Na₂SO₄), filtered and concentrated under reducedpressure. The resulting white solid was purified by flash LC (silicagel, gradient hex-EtOAc 4:1 to 1:1) to afford the title compound as awhite solid (700 mg, 73%, mixture of diastereomers).

¹H NMR (300 MHz, CDCl₃) δ 0.05 (s, 3H), 0.08 (s, 3H), 0.09 (s, 3H),0.68-1.05 (18H, m), 0.87 (9H, s), 1.05-1.85 (12H, m), 1.42 (9H, s),1.85-2.10 (3H, m), 2.11-2.31 (2H, m), 2.32-2.46 (2H, m), 2.47-2.60 (m,2H), 3.34-3.90 (2H, m), 3.93-4.30 (2H, m), 4.50-4.89 (6H, m), 4.90-5.12(2H, m), 5.07 (d, J=12.2, 2H), 5.18 (d, J=12.2, 2H), 5.60 (1H, d,J=9.7), 5.67 (1H, d, J=10.2), 5.89 (1H, d, J=11.2), 6.56 (1H, d, J=7.3),6.70 (1H, d, J=8.3), 6.76 (1H, d, J=6.8), 6.94 (d, J=6.8, 1H), 7.01-7.19(m, 1H), 7.32 (bs, 5H), 8.17 (1H, d, J=7.8), 8.28 (d, J=7.8, 1H).

ESI-MS Calcd for C₄₅H₇₉N₄O₉Si: 844.54. Found (m/z): 845.5 (M+H)⁺.

Example 22 Synthesis of Boc-Ist(TBDMS)-Hiv-Leu-Pro-OBn (SHPLA2)

Following the procedure described for the synthesis of SAPLA2, startingfrom SHPLA4 (850 mg, 2.0 mmol) and Cl (935 mg, 2.4 mmol) the titlecompound was obtained (1.53 g, 97%) after purification by flash LC(silica, gradient hex-EtOAc 3:1 to 2:1). Rf=0.63 (hex-EtOAc 2:1).

¹H NMR (300 MHz, CDCl₃) mixture of BocNH rotamers: δ 0.04 (s, 3H), 0.06(s, 3H), 0.88 (s, 9H), 0.78-1.04 (m, 18H), 1.10-2.80 (m, 11H), 1.44 (s,9H), 1.46 (s, 9H), 3.57 (m, 2H), 3.74 (m, 1H), 3.85 (m, 1H), 4.03 (m,1H), 4.23 (d, J=4.8, 1H), 4.48 (m, 1H), 4.85 (m, 1H), 4.90 (d, J=10,1H), 5.05 (m, 1H), 5.20 (d, J=10, 1H), 5.23 (d, J=10, 1H), 6.64 (d,J=6.4, 1H), 6.88 (d, J=8.6, 1H), 7.32 (m, 5H), 8.54 (d, J=8.3, 1H).

¹³C NMR (75 MHz, CDCl₃) δ−5.14, −4.58, 11.84, 12.97, 17.78, 17.92,18.98, 21.05, 23.11, 23.49, 25.61, 26.92, 28.36, 28.70, 30.12, 33.68,38.72, 42.86, 46.51, 48.18, 58.67, 60.24, 66.52, 71.14, 79.40, 82.66,127.96, 128.01, 128.33, 135.36, 157.30, 169.92, 171.10, 171.69, 171.97.

ESI-MS: Calcd for C₄₂H₇₁N₃O₉: 789.50. Found 790.5 (M+H)⁺.

Example 23 Synthesis of Boc-Ist(TBDMS)-Val-Leu-Pro-OBn (SVPLA2)

Following the procedure described for the synthesis of SNPLA2, startingfrom SVPLA3 (chlorhydrate) (1.2 g, 2.64 mmol), C1 (1.23 g, 3.17 mmol),DCC (654 mg, 3.17 mmol), HOBt (464 mg, 3.43 mmol), NMM (0.35 ml) and DCM(6 ml). The title compound was obtained as a white solid (1.87 g, 89%)after purification by flash LC (silica gel, gradient hex-EtOAc 3:1 to2:1).

¹H NMR (500 MHz, CDCl₃) δ 0.07 (bs, 6H), 0.81-0.96 (m, 27H), 1.11-1.38(m, 3H), 1.39-1.47 (bs, 7H), 1.51 (bs, 3H), 1.58-1.70 (m, 3H), 1.70-1.84(m, 1H), 1.86-2.60 (m, 4H), 2.28-2.58 (m, 2H), 3.58-3.62 (m, 1H),3.62-3.73 (m, 1H), 3.73-3.90 (m, 1H), 4.05-4.12 (m, 1H), 4.13-4.19 (m,1H), 4.19-4.23 (m, 1H), 4.49-4.54 (m, 1H), 4.77-5.06 (m, 2H), 5.18 (d,J=12.3, 1H), 5.55 (bs, 1H), 6.44-6.61 (m, 2H), 7.30-7.35 (m, 5H),7.94-7.98 (m, 1H).

ESI-MS Calcd for C₄₂H₇₂N₄O₈Si: 788.51. Found (m/z): 789.5 (M+H)⁺.

Example 24 Synthesis of Boc-Ist(TBDMS)-(Me)Val-Leu-Pro-OBn (SMPLA2)

To a flask containing SMPLA3 (chlorhydrate) (176 mg, 0.38 mmol), in DCM(2 ml, anh) at 0° C., NMM (41 □l, 0.38 mmol) was added. After 15 min, C1(176 mg, 0.46 mmol), and DCC (93 mg, 0.46 mmol) were added in portions.The flask was allowed to warm to room temperature and stirring wascontinued overnight. The reaction mixture was partitioned between DCM(10 ml) and aq KHSO₄ (2×5 ml, 10%). The organic phase was washedsuccessively with aq. NaHCO₃ (2×5 ml, sat), brine (5 ml), dried(Na₂SO₄), filtered and concentrated under reduced pressure. Theresulting white solid was purified by flash LC (silica, gradientHex-EtOAc 3:1 to 2:1) to give SMPLA2 (127 mg, 42%) as a white solid.Rf=0.51 (Hex-EtOAc 1:1).

¹H NMR (300 MHz, CDCl₃) δ−0.08 (s, 3H), 0.05 (s, 3H), 0.75-0.86 (m,27H), 1.00-1.43 (m, 11H), 1.52-1.65 (m, 1H), 1.68-1.80 (m, 1H),1.83-2.01 (m, 3H), 2.08-2.24 (m, 2H), 2.40 (m, 2H), 2.87 (s, 3H),3.50-3.57 (m, 3H), 3.71-3.76 (m, 1H), 4.29 (m, 1H), 4.47-4.63 (m, 4H),5.01 (d, J=12.9, 1H), 5.11 (d, J=12.9, 1H), 6.41 (d, J=7.8, 0.5H), 6.62(d, J=7.8, 1H), 7.01 (d, J=7.8, 0.5H), 7.23-7.28 (m, 5H).

¹³C NMR (75 MHz, CDCl₃) δ 11.56, 13.57, 13.98, 17.83, 18.87, 19.46,21.77, 22.94, 23.11, 24.50, 24.71, 24.80, 25.64, 25.76, 25.97, 27.29,28.18, 28.75, 30.40, 34.18, 39.28, 40.85, 46.58, 48.61, 57.03, 58.65,62.26, 66.63, 69.21, 78.72, 127.92, 128.05, 128.11, 128.33, 135.42,155.91, 169.76, 170.48, 171.56, 172.01.

ESI-MS Calcd for C₄₃H₇₄N₄O₈Si: 802.53. Found (m/z): 825.5 (M+Na)⁺.

Example 25 Synthesis of Ist-Hip-Leu-Pro-OBn (SAPLA1)

To a solution containing SAPLA2 (19.32 g, 22.8 mmol) in anh. dioxane (78ml), a solution of hydrochloric acid in anhydrous dioxane (4.2 N, 220ml, 924 mmol) was added. The resulting solution was stirred at 21° C.for 4.30 h or until complete disappearance of the starting material(TLC). Then, the solution was concentrated under reduced pressure. Theresidue was dissolved in DCM (25 ml) and concentrated to remove residualHCl. The resulting residue was dried under vacuum until completeelimination of free HCl (3 h) to give 17.3 g of SAPLA1 (15.1 g, quant)as a white foam (mixture of diasteromers).

Example 26 Synthesis of Ist-Aip-Leu-Pro-OBn (SNPLA1)

Following the procedure described for synthesis of SAPLA1, starting fromSNPLA2 (700 mg, 0.82 mmol), the title compound was obtained as a whitesolid (545 mg, quant.) after precipitation with Et₂O (mixture ofdiasteromers).

¹H NMR (300 MHz, CDCl₃) δ 0.86-1.04 (m, 18H), 1.02-1.22 (m, 3H),1.23-1.58 (m, 5H), 1.60-1.80 (m, 2H), 1.82-2.01 (m, 3H), 2.24 (m, 2H),2.40-2.85 (m, 2H), 3.24 (m, 1H), 3.45 (m, 1H), 3.60 (m, 1H), 3.70-4.05(m, 2H), 4.46 (m, 2H), 4.47-4.75 (m, 2H), 5.10 (bs, 2H), 7.34 (bs, 5H),7.98 (bs, 1H), 8.10 (bs, 1H).

ESI-MS Calcd for C₃₄H₅₄N₄O₇:630.40. Found (m/z): 631.4 (M+H)⁺.

Example 27 Synthesis of Ist-Hiv-Leu-Pro-OBn (SHPLA1)

Following the procedure described for synthesis of SAPLA1, starting fromSHPLA2 (1.53 g, 1.94 mmol), the title compound (1.12 g, quant.) wasobtained as a white solid after precipitation with Et₂O.

¹H NMR (300 MHz, CDCl₃) δ 0.86-1.04 (m, 18H), 1.10-1.22 (m, 3H), 1.42(m, 2H), 1.70 (m, 2H), 1.97 (m, 3H), 2.24 (m, 2H), 2.83 (m, 1H), 2.97(m, 1H), 3.34 (m, 1H), 3.61 (m, 1H), 3.75 (m, 1H), 3.90 (m, 1H), 4.56(m, 2H), 4.75 (m, 1H), 5.04 (d, J=11, 1H), 5.18 (d, J=11, 1H), 7.34 (bs,5H), 8.21 (bs, 3H).

ESI-MS Calcd for C₃₁H₄₉N₃O₇: 575.36. Found (m/z): 576.3 (M+H)⁺.

Example 28 Synthesis of Ist-Val-Leu-Pro-OBn (SVPLA1)

Following the procedure described for synthesis of SAPLA1, starting fromSVPLA2 (1.87 g, 2.36 mmol), the title compound (1.40 g, quant.) wasobtained as a white solid after precipitation with Et₂O.

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.01 (m, 15H), 1.01-1.10 (m, 3H),1.20-1.79 (m, 5H), 1.81-2.05 (m, 4H), 2.05-2.15 (m, 2H), 2.50-2.68 (m,2H), 2.82-3.1 (m, 1H), 3.20-3.35 (m, 1H), 3.50-3.70 (m, 1H), 3.80-3.90(m, 1H), 4.18-4.30 (m, 1H), 4.35-4.45 (m, 1H), 4.45-4.55 (m, 1H),4.60-4.70 (m, 1H), 5.02 (d, J=12.3, 1H), 5.15 (d, J=12.3, 1H), 7.28-7.38(m, 5H), 7.5 (bs, 1H), 7.9 (bs, 3H), 8.15 (bs, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 10.81, 14.67, 18.29, 19.32, 21.48, 23.14,24.48, 24.76, 25.81, 30.29, 33.41, 40.31, 46.96, 49.35, 59.14, 59.94,60.67, 66.94, 128.11, 128.32, 128.54, 135.32, 171.58, 171.74, 171.80,172.57.

ESI-MS Calcd for C₃₁H₅₀N₄O₆: 574.35. Found (m/z): 575.3 (M+H)⁺.

Example 29 N-tert-Butyloxycarbonylthreonine Phenacyl ester (D3)

To a stirred suspension of Boc-Thr-OH (21.91 g, 0.1 mol) in EtOAc (200ml) at 0° C., TEA (14 ml, 0.1 mol) and bromoacetophenone (19.0 g, 0.1mol) were added. The reaction mixture was allowed to warm to 20° C.,stirred for 2 days and then diluted with EtOAc (500 ml). After washingsuccessively with aq HCl (200 ml, 0.1 N), H₂O (100 ml), aq NaHCO₃ (200ml, 1 N) and brine (200 ml), drying (Na₂SO₄), filtered and concentratedin vacuo, the residue was triturated with Et₂O and filtered. Theresulting solid was dried in the dark to yield D3 (28.6 g, 85%). Rf=0.55(hex-EtOAc 1:1, silica); M.p.=114.2° C.; [α]_(D) ²²−29.4 (c 2, EtOAc).

¹H NMR (300 MHz, CDCl₃): δ=1.31 (d, J=6.6, 3H), 1.46 (s, 9H), 3.77 (brd, OH), 4.44 (dd, J=9.6, 1H), 4.6 (q, 1H), 5.34 (d, J=16.5, 1H) 5.37 (brd, OH) 5.68 (d, J=16.8, 1H), 7.51 (t, 2H), 7.65 (t, 1H), 7.92 (dd, 2H).

Example 30 N-Benzyloxycarbonyl-N,O-Dimethyl-L-tyrosine (E1)

To a stirred solution of Z-Tyr-OH (63.24 g, 200 mmol) in THF (900 ml) at0° C. was added finely powdered KOH (112.72 g, 2 mol) in portions,followed by the addition of tetrabutylammonium hydrogen sulfate (6.36 g,10% by weight). Then, dimethyl sulfate (127.2 ml, 1.33 mol) was addeddropwise over 30 min, while maintaining the reaction mixture below 4° C.The reaction was stirred for an additional 30 min and H₂O (950 ml) wasadded. After stirring 5 h at O C, the reaction was diluted with ether(1500 ml), the aqueous layer was separated, and the organic layer wasextracted with aq NaHCO₃ (2×500 ml, sat). The combined aqueous layerswere acidified to pH 1 with aq 1M KHSO₄, and extracted with EtOAc (5×500ml). The organic layers were combined, dried (Na₂SO₄), filtered andconcentrated. The residue was precipitated with ethyl ether and filteredto give E1 as a white solid (53.85 g, 78%).

[α]_(D) ²²−57.16 (c 2.23 CHCl₃) (lit [α]_(D)−48 (c=2.23 CHCl₃). JACS,112, 21, 1990).

Example 31O-(Benzyloxycarbonyl-N,O-dimethyl-L-tyrosyl)-N-tert-Butyloxycarbonyl-L-threoninePhenacyl ester (D2)

To a solution of D3 (33.72 g, 100 mmol) in DCM at 0° C., DMAP (3.66 g,30 mmol), and E1 (34.33 g, 100 mmol) were added. After stirring 10 minat 0° C., DCC (22.7 g, 110 mmol) was added. The reaction mixture wasallowed to warm to room temperature and stirred overnight. Then themixture was filtered and the filtrate concentrated to dryness. Theresidue was chased with ACN (100 ml), filtered again and the filtratewas concentrated. The residue was dissolved in EtOAc (200 ml) andpartitioned successively between aq KHSO₄ (100 ml, 10%), aq NaHCO₃ (100ml, sat.) and brine (100 ml). The organic phase was dried (Na₂SO₄),filtered and concentrated. The residue was purified by flash LC (silicagel, grad EtOAc-Hex 1:4 to 1:2) to yield D2 (65.5 g, 98%). [α]_(D)²²−39.56 (c 1.06 CHCl₃); Rf=0.55 (EtOAc:Hex 1:1).

Example 32O-(Benzyloxycarbonyl-N,O-dimethyl-L-tyrosyl)-N-tert-Butyloxycarbonyl-L-threonine(D1)

To a homogeneous solution of D2 (24.49 g, 38.4 mmol) in aq AcOH (211 ml,90%) at 0° C., powdered Zn was added (18.65 g, 288.3 mmol). Theresulting mixture was stirred at 0° C. for 3 h until disappearance ofthe starting material (followed by TLC). The reaction mixture wasfiltered over celite and washing with EtOAc (200 ml). The filtrate wasconcentrated at reduced pressure and the residue was chased with Et₂O(200 ml) and filtered. The filtrate was successively partitioned betweenaq KHSO₄ (100 ml, 10%) and brine (100 ml). The organic phase was dried(Na₂SO₄) and concentrated to give an oil which was purified by flash LC(Lichroprep RPC18, ACN:H₂O 1:1 (800 ml, then 7:3 (600 ml)] to yield D1(15.53 g, 74%) as a white solid. [α]_(D) ²⁴−27.6 (c 2.187, DCM); lit[α]_(D)−20.5 (c 2, DCM). JOC, 62, 2, 1997. Rf=0.58 [ACN/H₂O (7:3)].

IR (film, DCM) ν 3400, 3050, 2900, 1715, 1613, 1514, 1456, 1402, 1368,1248, 1165, 1061, 1036 cm⁻¹.

¹H NMR (200 MHz, CDCl₃) δ 1.29 (d, J=6.5, 3H), 1.45 (s, 9H), 2.74 (s,3H), 2.75 (s, 3H), 2.76-3.31 (m, 2H), 3.77 (s, 3H), 4.42-4.52 (m, 1H),4.66-4.83 (m, 1H), 5.01-5.16 (m, 2H), 5.30-5.53 (m, 2H), 6.72-6.81 (m,2H), 6.95-7.09 (m, 2H), 7.35 (bs, 5H).

¹³C NMR (75 MHz, CDCl₃) δ 16.44, 16.82, 28.23, 31.71, 31.97, 33.82,33.68, 55.14, 56.87, 56.75, 60.39, 60.58, 67.51, 67.76, 71.83, 72.47,80.40, 113.91, 127.59, 128.69, 129.77, 136.42, 156.00, 156.19, 156.71,158.31, 159.47, 169.78.

m/z (FAB) 567.1 [(M+Na)⁺, 46], 545.1 [(M+H)⁺, 7], 445.1 (100); m/z(FABHRMS) 567.233 280, C₂₈H₃₅N₂O requires (M+Na)⁺ 567.231 851.

Example 33 Synthesis of Boc-Thr(Z-N(Me)-O(Me)-Tyr)-Ist-Hip-Leu-Pro-OBn(SAPL7)

To flask containing HBTU (9.079 g, 23.9 mmol), HOBt (3.490 g, 22.8mmol), SAPLA1 (15.258 g, 22.8 mmol) and D1 (12.417 g, 22.8 mmol), asolution of anh DCM (296 mL) and anh DMF (148 mL) were cannulated underAr at −5° C. After 5 min of stirring, DIPEA (15.9 mL, 91.2 mmol) wasadded dropwise by syringe, while maintaining the temperature <−5° C. Theresulting reaction mixture was stirred for 21 h at −5° C. MTBE (300 mL)and KHSO₄ (200 mL, 10%) were added, and the resulting mixture wasfiltered off and concentrated up to 300 mL. Additional MTBE (200 mL) wasadded, the layers were separated, and the organic phase was treatedsequentially with aq. KHSO₄ (200 ml, 10%), brine (200 ml), aq. NaHSO₄(200 ml, sat.) and rinse with brine (200 ml). The organic phase wasdried (Na₂SO₄) and concentrated under reduced pressure to afford ayellow oil (30 g). The oil was dissolved in MTBE and treated with hexwhile stirring. Solid precipitated and more hex was added. The solid wasfiltered to yield SAPL7 (18.33 g, 69% yield) as a white solid. Thisproduct is a mixture of two diastereomers. Rf=0.80 and 0.59 (hex:EtOAc1:2).

IR (film, DCM) ν 3350, 2961, 2927, 2893, 1744, 1688, 1638, 1514, 1454,1368, 1304, 1248, 1171, 1067, 1036 cm⁻¹.

¹H NMR (500 MHz, CDCl₃) δ 0.74-0.92 (m, 18H), 1.05-1.15 (m, 2H),1.18-1.20 (m, 2H), 1.23 (d, J=6.8, 3H), 1.25 (d, J=6.8, 3H), 1.29 (d,J=6.9, 3H), 1.42 (s, 9H), 1.45 (s, 9H), 1.50-1.66 (m, 3H), 1.89-2.02 (m,4H), 2.17-2.25 (m, 2H), 2.37-2.42 (m, 1H), 2.81 (s, 3H), 2.88 (s, 3H),2.91 (s, 3H), 2.95 (s, 3H), 2.84-2.93 (m, 2H), 3.17-3.25 (m, 1H),3.53-3.59 (m, 1H), 3.75 (s, 3H), 3.88-3.98 (m, 4H), 4.49 (d, J=3.1, 1H),4.51 (d, J=3.1, 1H), 4.53-4.57 (m, 1H), 4.68-4.72 (m, 1H), 4.96-4.99 (m,1H), 5.02-5.33 (m, 4H), 5.02 (d, J=3.2, 1H), 5.23 (d, J=3.1, 1H),5.26-5.33 (m, 1H), 5.47 (1d, J=9.5, 1H), 6.74 (d, J=7.8, 2H), 6.77 (d,J=7.7, 2H), 7.08 (d, J=7.7, 2H), 7.17 (d, J=7.5, 1H), 7.21 (d, J=9.5,1H), 7.23-7.36 (m, 10H), 7.75 (d, J=7.9, 1H), 7.79 (d, J=8.2, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.95, 13.27, 15.16, 16.47, 17.33, 18.79,21.28, 23.65, 24.65, 24.72, 27.09, 28.08, 28.93, 31.20, 31.32, 33.62,33.98, 38.38, 41.01, 47.12, 49.38, 54.96, 55.17, 57.89, 58.83, 60.01,60.16, 67.18, 71.05, 71.32, 80.34, 81.24, 113.89, 127.51, 128.59,129.69, 129.77, 135.52, 136.77, 156.93, 158.27, 169.87, 170.62, 171.15,171.85, 172.39, 204.88.

m/z(FAB) 1181.2 [(M+Na)⁺, 20], 1159.2 [(M+H)⁺, 80], 1059.2 (100). Anal.Calcd for C₆₂H₈₇N₅O₁₆: C, 64.30; H, 7.52; N, 6.05. Found: C, 64.14; H,7.66; N, 5.95

Example 34 Synthesis of Boc-Thr(Z-N(Me)-O(Me)-Tyr)-Ist-Aip-Leu-Pro-OBn(SNPL7)

Following the procedure described for synthesis of SAPL7, starting fromSNPLA1 (150 mg, 0.22 mmol) and D1 (122 mg, 0.22 mmol), the titlecompound was obtained as a white solid (130 mg, 51%) after purificationby flash LC (silica gel, gradient hex-EtOAc 2:1 to 1:3) (mixture ofdiastereomers).

¹H NMR (300 MHz, CDCl₃) δ 0.74-1.03 (m, 18H), 1.16-1.37 (m, 10H), 1.45(s, 9H), 1.68 (m, 3H), 1.99 (m, 4H), 2.22 (m, 2H), 2.48 (m, 1H), 2.82(s, 3H), 2.84-3.10 (m, 2H), 3.19 (m, 1H), 3.51-3.69 (m, 2H), 3.75 (s,3H), 3.72-4.02 (m, 3H), 4.18 (m, 1H), 4.50-4.73 (m, 4H), 5.00-5.27 (m,5H), 5.49 (m, 2H), 6.54 (d, J=9.2, 1H), 6.78 (d, J=6.8, 2H), 7.02 (d,J=6.8, 2H), 7.18 (m, 1H), 7.23-7.36 (m, 10H), 7.52 (d, J=6.8, 1H).

ESI-MS Calcd for C₆₂H₈₈N₆O₁₅: 1156.63. Found 1158.3 (M+H)⁺.

Example 35 Synthesis of Boc-Thr(Z-N(Me)-O(Me)-Tyr)-Ist-Hiv-Leu-Pro-OBn(SHPL7)

Following the procedure described for synthesis of SAPL7, starting fromSHPLA1 (1.12 g, 1.94 mmol) and SAPLD1 (544.6 mg, 1.94 mmol), the titlecompound (1.045 g, 61%) was obtained as a white solid after purificationby flash LC (silica gel, gradient hex-EtOAc 1:1 to 1:2). Rf=0.46(hex-EtOAc 1:2).

¹H NMR (300 MHz, CDCl₃) δ 0.74-1.02 (m, 18H), 1.20 (m, 5H), 1.40 (m,3H), 1.46 (s, 9H), 1.62 (m, 2H), 1.82-2.20 (m, 3H), 2.20 (m, 2H), 2.50(m, 1H), 2.78 (s, 3H), 2.90 (m, 1H), 3.20 (m, 1H), 3.58 (m, 1H), 3.67(s, 3H), 3.79 (m, 1H), 3.88 (m, 1H), 4.06 (m, 2H), 4.40 (m, 2H), 4.82(m, 2H), 4.94 (m, 1H), 4.98 (m, 1H), 5.08 (m, 1H), 5.28 (m, 3H), 5.56(d, J=6.2, 1H), 6.84 (d, J=8.3, 2H), 6.98 (d, J=6.5, 1H), 7.07 (d,J=8.3, 2H), 7.34 (m, 10H), 7.52 (d, J=6.2, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.27, 13.77, 13.95, 16.75, 17.46, 18.31,20.78, 20.96, 23.09, 24.35, 24.53, 26.74, 27.93, 28.63, 30.17, 33.76,39.06, 40.01, 46.69, 48.17, 54.89, 57.04, 57.68, 58.65, 60.20, 60.60,66.74, 67.08, 68.31, 70.30, 78.49, 79.90, 113.62, 127.36, 127.70,128.14, 128.18, 128.31, 129.64, 135.19, 136.31, 155.86, 156.61, 158.09,169.77, 170.70, 171.06, 171.17, 171.78.

ESI-MS Calcd for C₅₉H₈₃N₅O₁₅: 1101.59. Found 1102.7 (M+H)⁺.

Example 36 Synthesis of Boc-Thr(ZN(Me)-O(Me)-Tyr)-Ist-Val-Leu-Pro-Bn(SVPL7)

Following the procedure described for synthesis of SAPL7, starting fromSVPLA1 (1.44 g, 2.37 mmol) and D1 (1.29 g, 2.37 mmol), the titlecompound (1.96 g, 75%) was obtained as a white solid after purificationby flash LC (silica gel, gradient hex-EtOAc 2:1 to 1:3). Rf=0.56(EtOAc).

¹H NMR (300 MHz, CDCl₃): δ 0.83-0.95 (m, 15H), 1.0-1.22 (m, 4H),1.23-1.44 (m, 9H), 1.60-1.65 (m, 1H), 1.87-2.01 (m, 4H), 2.09-2.20 (m,3H), 2.77 (bs, 8H), 2.84-3.01 (m, 1H), 3.17-3.24 (m, 1H), 3.51-3.60 (m,1H), 3.73 (s, 3H), 3.80-3.90 (m, 2H), 4.03-4.15 (m, 1H), 4.25-4.40 (m,2H), 4.40-4.52 (m, 1H), 4.70-4.80 (m, 2H), 5.00-5.26 (m, 4H), 5.34-5.36(m, 1H), 5.58 (m, 1H), 6.75 (d, 2H, J=7.8), 6.96-7.09 (m, 1H), 7.04 (d,2H, J=8.1), 7.04-7.12 (m, 1H), 7.16-7.20 (m, 1H), 7.18-7.30 (m, 10H).

¹³C NMR (75 MHz, CDCl₃) δ 11.43, 13.63, 17.17, 18.35, 19.21, 21.40,23.23, 24.49, 24.67, 26.87, 28.10, 28.79, 30.48, 32.11, 33.84, 38.47,40.37, 41.22, 46.80, 48.61, 55.04, 56.88, 57.95, 58.75, 59.25, 60.82,66.87, 67.33, 69.40, 70.50, 76.44, 80.45, 113.58, 127.51, 127.74,127.88, 128.10, 128.25, 128.33, 128.46, 128.72, 129.64, 129.77, 135.35,136.37, 156.77, 158.29, 169.83, 170.57, 171.3, 171.4, 172.81.

ESI-MS Calcd for C₅₉H₈₄N₆O₁₄: 1100.60. Found (m/z): 1101.7 (M+H)⁺.

Example 37 Synthesis of Boc-Thr(N(Me)-O(Me)-Tyr)-Ist-Hip-Leu-Pro-OH(SAPL6)

To a solution of SAPL7 (18.33 g, 15.8 mmol) in THF (free of stabilizer,500 mL) degassed and purged with argon, Pd(OH)₂—C (20% Pd, 7.33 g, 40%w/w). The mixture was stirred under H₂ (1 atm) for 20 h, then filteredover a 0.45 □ teflon filter and concentrated under reduced pressure togive a white solid. Toluene (30 mL) was added, and concentrated againunder reduced pressure and high vacuo to give SAPL6 (14.78 g, quant) asa white solid.

¹H NMR (500 MHz, CDCl₃) δ 0.79-1.08 (m, 18H), 1.09-1.18 (m, 3H), 1.26(bs, 3H), 1.29 (d, J=7.1, 3H), 1.47 (s, 9H), 1.50-1.66 (m, 3H),1.84-1.94 (m, 1H), 1.90-2.28 (m, 4H), 2.35-2.50 (m, 4H), 2.30-2.35 (m,1H), 2.44-3.18 (m, 4H), 2.60 (m, 3H), 3.53-3.61 (m, 1H), 3.77 (s, 3H),3.88-4.07 (m, 4H), 4.12-4.72 (m, 4H), 5.18-5.24 (m, 1H), 5.24 (bs, 1H),6.84 (d, J=7.9, 2H), 7.08 (d, J=8.0, 2H), 7.13 (d, J=8.2, 1H), 7.18 (d,J=8.2, 1H), 7.62-7.68 (bs, 1H).

m/z (FAB) 972.7 [(M+K)⁺, 33], 934.9 (M)⁺, 100].

Example 38 Synthesis of Boc-Thr(N(NMe)-O(Me-Tyr)-Ist-Aip-Leu-Pro-OH(SNPL6)

To a solution of SNPL7 (130 mg, 0.11 mmol) in a mixture IPA:H2O (2:1, 4ml:2 ml) degassed and purged with argon, Pd(OH)₂—C (20% Pd, 45 mg, 35%w/w). The mixture was stirred under H₂ (1 atm) for 20 h, then filteredover a 0.45 □ teflon filter and concentrated under reduced pressure togive a white solid. IPA (10 ml) was added, and concentrated again underreduced pressure and high vacuo to give SNPL6 (100 mg, quant) as a whitesolid.

ESI-MS Calcd for C₄₇H₇₆N₆O₁₃: 932.55. Found 934.0 (M+H)⁺.

Example 39 Synthesis of Boc-Thr(N(Me)-O(Me)-Tyr)-Ist-Hiv-Leu-Pro-OH(SHPL6)

Following the procedure described for synthesis of SAPL6, starting fromSHPL7 (1.045 g, 0.95 mmol). The title compound (825 g, 99%) was obtainedas a white solid.

ESI-MS Calcd for C₄₄H₇₁N₅O₁₃: 877.50. Found 878.5 (M+H)⁺.

Example 40 Synthesis of Boc-Thr(N(Me)-O(Me)-Tyr)-Ist-Val-Leu-Pro-OH(SVPL6)

Following the procedure described for synthesis of SAPL6, starting fromSVPL7 (250 mg, 0.23 mmol). The title compound (195 mg, 97%) was obtainedas a white solid.

ESI-MS Calcd for C₄₄H₇₂N₆O₁₂: 876.56. Found (m/z): 877.5 (M+H)⁺.

Example 41 Synthesis ofCyclo-N(Me)-O(Me)-Tyr-O-(Boc-Thr)-Ist-Hip-Leu-Pro (SAPL5)

In a cooled (−5° C.) 5 L reactor fitted with mechanical stirrercontaining ACN (3.2 L), HATU (14.436 g, 37.9 mmol) and HOAt (5.254 g,38.6 mmol) were added under Ar while stirring. SAPL6 (14.77 g, 15.8mmol) dissolved in ACN (500 ml) was added. NMM (3.65 ml, 33.18 mmol) wasadded dropwise by syringe while maintaining the temperature below −5° C.The resulting reaction mixture was allowed to reach room temperature andwas stirred for 20 h. The solvent was evaporated under reduced pressure.The crude was chased with EtOAc (500 ml) and the solution was filteredoff to remove precipitate. The solution was washed successively with aq.KHSO₄ (2×500 ml), brine (500 ml), aq. NaHCO₃ (500 ml, sat.) and rinsewith brine (500 ml). The organic phase was dried (Na₂SO₄) andconcentrated under reduced pressure to afford a yellow solid (17.52 g)that was purified by flash (silica gel, grad hex:EtOAc 3:1 to 1:1) togive SAPL5 (9.83 g, 68%) as a white solid. Rf=0.60 (Hex:EtOAc 1:3).[α]_(D)−209.4 (c 0.3, CHCl₃).

IR (film, DCM) ν 3343, 2961, 2927, 2893, 1734, 1640, 1514, 1454, 1368,1302, 1248, 1167, 1018 cm⁻¹.

¹H NMR (500 MHz, CDCl₃) δ 0.78 (d, J=7.1, 3H), 0.85 (d, J=7.0, 3H), 0.87(d, J=7.0, 3H), 0.89-0.93 (m, 9H), 1.10-1.20 (m, 1H), 1.20 (d, J=6.4,3H), 1.30 (d, J=6.9, 3H), 1.36 (m, 2H), 1.40 (m, 2H), 1.44 (s, 9H),1.48-1.72 (m, 2H), 1.72-1.78 (m, 1H), 1.83-1.88 (m, 1H), 2.01-2.17 (m,3H), 2.27-2.29 (m, 1H), 2.47-2.53 (m, 1H), 2.53 (s, 3H), 2.93 (bs, 1H),3.14-3.19 (m, 2H), 3.34-3.37 (dd, J₁=14.8, J₂=4.1, 1H), 3.54-3.56 (dd,J₁=10.5, J₂=4.1, 1H), 3.58-3.63 (m, 1H), 3.68-3.72 (m, 1H), 3.78 (s,3H), 3.94-3.98 (m, 1H), 3.98 (q, J=7.5, 1H), 4.07-4.11 (3d, J=3.8, 1H),4.57-4.61 (m, 2H), 4.77-4.81 (m, 1H), 4.97-4.98 (q, J=3.5, 1H), 5.02 (d,J=10.5, 1H), 5.18 (d, J=4.2, 1H), 6.81 (d, J=8.5, 2H), 7.05 (d, J=8.5,2H), 7.19 (d, J=10.2, 1H), 7.64 (d, J=10.1, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.56, 14.68, 14.97, 15.27, 16.61, 18.45,20.64, 23.50, 24.71, 24.78, 26.92, 27.73, 27.94, 31.55, 33.94, 33.94,38.27, 38.52, 40.64, 46.86, 49.54, 49.65, 55.16, 55.19, 55.84, 57.12,65.96, 67.30, 71.00, 80.27, 81.41, 114.02, 130.22, 158.53, 168.30,169.31, 170.12, 170.29, 171.20, 172.38, 204.51.

m/z (FAB) 938.9 [(M+Na)⁺, 55], 916.9 [(M+H)⁺, 100]; m/z (FABHRMS)916.532 120, C₄₇H₇₃N₅O₁₃, requires (M+H)⁺=916.528 300.

Example 42 Synthesis ofCyclo-N(Me)-O(Me)-Tyr-O-(Boc-Thr)-Ist-Aip-Leu-Pro (SNPL5)

Following the procedure described for synthesis of SAPL5, starting fromSNPL6 (100 mg, 0.11 mmol), the title compound (40 mg, 57%) was obtainedas a white solid after flash LC (silica gel, EtOAc:hex 4:1 to EtOAcneat). Rf=0.4 (EtOAc).

¹H NMR (300 MHz, CDCl₃) δ 0.76 (d, J=6.8, 3H), 0.83-0.96 (m, 15H),1.10-1.20 (m, 1H), 1.25 (d, J=6.4, 3H), 1.27 (d, J=6.3, 3H), 1.32 (d,J=6.8, 3H), 1.41 (m, 2H), 1.44 (s, 9H), 1.50-1.70 (m, 2H), 1.99-2.31 (m,5H), 2.61 (s, 3H), 2.91-3.04 (m, 1H), 3.11-3.37 (m, 2H), 3.48-3.64 (m,3H), 3.69-3.81 (m, 1H), 3.80 (s, 3H), 4.18 (m, 2H), 4.46-4.67 (m, 3H),4.81 (t, J=10.7, 1H), 5.01 (m, 1H), 6.85 (d, J=8.3, 2H), 7.07 (d, J=8.3,2H), 7.33 (d, J=8.7, 1H), 7.65 (d, J=9.2, 1H), 7.86 (d, J=10.7, 1H).

ESI-MS Calcd for C₄₇H₇₄N₆O₁₂ 914.54. Found m/z 915.5 (M+H)⁺.

Example 43 Synthesis ofCyclo-N(Me)-O(Me)-Tyr-O-(Boc-Thr)-Ist-Hiv-Leu-Pro (SHPL5)

Following the procedure described for synthesis of SAPL5, starting fromSHPL6 (2.45 g, 2.78 mmol), the title compound (2.1 g, 88%) was obtainedas a white solid after cristalization of DCM/n-heptane (1:3). Rf=0.33(hex-EtOAc 1:3).

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.04 (m, 18H), 1.19 (m, 5H), 1.41 (s,9H), 1.42 (m, 2H), 1.63 (m, 1H), 1.77 (m, 1H), 1.90 (m, 1H), 2.00-2.22(m, 3H), 2.44 (m, 1H), 2.58 (s, 3H), 2.95 (m, 1H), 3.14 (m, 1H), 3.26(m, 1H), 3.36 (m, 1H), 3.58 (m, 1H), 3.68 (m, 2H), 3.78 (s, 3H), 3.96(m, 1H), 4.12 (m, 1H), 4.30 (m, 1H), 4.61 (m, 1H), 4.87 (m, 1H), 4.94(m, 1H), 5.03 (m, 1H), 6.84 (d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.54(d, J=7.3, 1H), 7.69 (d, J=6.4, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.38, 14.57, 15.15, 18.11, 18.45, 20.67,23.40, 24.70, 26.91, 27.89, 30.20, 33.57, 33.90, 38.51, 39.07, 46.62,48.13, 55.16, 56.05, 56.23, 56.94, 65.67, 68.60, 71.13, 79.41, 80.01,113.97, 129.69, 130.25, 155.75, 158.49, 168.51, 169.57, 170.24, 170.94,171.06, 173.59.

ESI-MS: Calcd for C₄₄H₆₉N₅O₁₂ 859.49. Found m/z 860.4 (M+H)⁺.

Example 44 Synthesis ofCyclo-N(Me)-O(Me)-Tyr-O-(Boc-Thr)-Ist-Val-Leu-Pro (SVPL5)

Following the procedure described for synthesis of SAPL5, starting fromSVPL6 (90 mg, 0.1 mmol), 30 mg (35%) of the title compound was obtainedas a white solid after purification by flash LC (silica, gradienthex-EtOAc from 1:4 to 1:10). Rf=0.35 (EtOAc).

¹H NMR (300 MHz, CDCl₃): δ. 0.85-1.00 (m, 18H), 1.14-1.38 (m, 8H), 1.44(s, 9H), 1.57 (m, 2H), 1.76-1.95 (m, 2H), 2.01-2.21 (m, 2H), 2.33 (dd,J₁=7.3, J₂=14.7, 1H), 2.53 (m, 1H), 2.57 (s, 3H), 3.17 (dd, J₁=10.7,J₂=14.7, 1H), 3.35 (dd, J₁=4.4, J₂=14.2, 1H), 3.56 (dd, J₁=3.9, J₂=10.3,1H), 3.59-3.77 (m, 4H), 3.78 (s, 3H), 4.06 (dt, J₁=3.9, J₂=9.3, 1H),4.33 (dd, J₁=2.9, J₂=9.3, 1H), 4.38 (dd, J₁=6.8, J₂=10.3, 1H), 4.58 (dd,J₁=5.4, J₂=7.3, 1H), 4.79 (t, J=10.3, 1H), 4.98 (d, J=9.3, 1H), 5.03(dd, J₁=2.4, J₂=6.3, 1H), 6.81 (bs, 1H), 6.84 (d, J=8.3, 2H), 7.08 (d,J=8.3, 2H), 7.24 (d, J=9.8, 1H), 7.54 (d, J=9.8, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.48, 14.50, 15.20, 18.86, 19.44, 21.03,23.64, 24.83, 24.96, 26.97, 28.01, 28.135, 30.21, 33.53, 33.96, 38.56,38.61, 41.05, 41.66, 46.84, 48.53, 55.24, 55.51, 56.31, 57.14, 59.75,65.85, 70.60, 80.52, 114.11, 129.75, 130.33, 156.77, 158.67, 168.53,170.21, 170.29, 170.73, 171.02, 174.34.

ESI-MS Calcd for C₄₄H₇₀N₆O₁₁: 858.51. Found (m/z): 859.5 (M+H)⁺.

Example 45 Synthesis of Cyclo-N(Me)-O(Me)-Tyr-O-(Thr)-Ist-Hip-Leu-Pro(SAPL4)

To a flask containing SAPL5 (8.79 g, 9.6 mmol) in anh. dioxane (93 mL),a solution of hydrochloric acid in anh. dioxane (5.3 N, 122 mL, 647mmol) was added. The resulting solution was stirred at room temperaturefor 8 h or until complete disappearance of the starting material. Whenthe reaction was completed, the solution was concentrated under reducedpressure. The residue was diluted with CHCl₃ (100 ml) and concentratedagain. The white foam crude was coevaporated with CHCl₃/hex to giveSAPL4 (8.17 g, 100% yield) as a white solid.

m/z (FAB) 838.3 [(M+Na)⁺, 28], 816.3 [(M+H)⁺, 100].

Example 46 Synthesis of Cyclo-N(Me)-O(Me)-Tyr-O-(Thr)-Ist-Aip-Leu-Pro(SNPL4)

Following the procedure described for the synthesis of SAPL4, startingfrom SNPL5 (40 mg, 43 μmol), the title compound (36 mg, quant.) wasobtained as a white solid after precipitation with Et₂O.

ESI-MS: Calcd for C₄₂H₆₅N₅O₁₁ 815.47. Found m/z 815.5 (M)⁺.

Example 47 Synthesis of Cyclo-N(Me)-O(Me)-Tyr-O-(Thr)-Ist-Hiv-Leu-Pro(SHPL4)

Following the procedure described for synthesis of SAPL4, starting fromSHPL5 (500 mg, 0.58 mmol). The title compound (440 mg, quant.) wasobtained as a white solid after precipitation with Et₂O.

ESI-MS: Calcd for C₃₉H₆₁N₅O₁₀ 759.44. Found m/z 760.4 (M+H)⁺.

Example 48 Synthesis of Cyclo-N(Me)-O(Me)-Tyr-O-(Thr)-Ist-Val-Leu-Pro(SVPL4)

Following the procedure described for synthesis of SAPL4, starting fromSVPL5 (25 mg, 29 □mol). The title compound (22 mg, quant.) was obtainedas a white solid after coevaporation with MTBE.

ESI-MS Calcd for C₃₉H₆₂N₆O₉: 758.5. Found (m/z): 759.5 [(M+H)]⁺.

Example 49 Z-N-Methyl-D-Leucine (H1)

Ref: Coggins, J. R.; Benoiton, N. L. Can. J. Chem 1971, 49, 1968.

To a stirred solution of Z-D-Leu-OH (10.32 g, 38.9 mmol) in anh. THF(120 mL) at 0° C. under Ar, Iodomethane (8.55 mL, 136.1 mmol) was addeddropwise by syringe. Then, sodium hydride (4.80 g, 120.6 mmol, 60%dispersion in mineral oil) was added in portions while maintaining thetemperature at 0° C. The reaction mixture was stirred at roomtemperature for 24 h. The solvent was eliminated under reduced pressureand the residue was dissolved in EtOAc (120 mL) and extracted with aq.NaHCO₃ (300 mL, sat). The aqueous phase was washed with EtOAc (2×100ml). The aqueous phase was cooled down, solid cytric acid was added upto pH 1-2, and the solution was extracted with EtOAc (4×250 mL), dried(Na₂SO₄), filtered and concentrated. The product was crystallized inEtOAc-Heptane (1:3) to obtain H1 (7.84 g, 72%) as a white cristallinesolid. Mp: 71-72° C. [α]_(D) ²⁵+23 (c 1, EtOH).

Example 50 Synthesis of Z-Didemnin A (SAPL3)

To a flask containing HATU (8.76 g, 23.0 mmol), HOAt (3.17 g, 23.1 mmol)SAPL4 (7.09 g, 15.8 mmol) and H1 (3.486 g, 12.5 mmol), anh. DCM (100 mL)and anh. DMF (50 mL) were added under Ar and the solution was stirred at−5° C. (ice bath). NMM (2.3 ml, 21.0 mmol) was added dropwise bysyringe, while maintaining the temperature at −5° C. The resultingmixture reaction was stirred at −5° C. for 2 h, then allowed to reachroom temperature for additional 14 h. The solvent was evaporated underreduced pressure. The crude was chased with EtOAc (100 ml) and thesolution was filtered off to remove some precipitate. The solution waswashed successively with aq. KHSO₄ (2×100 ml, 10%), brine (100 ml), aq.NaHCO₃ (100 ml, sat.) and rinse with brine (100 ml). The organic phasewas dried (Na₂SO₄) and concentrated under reduced pressure to afford ayellow solid that was purified by flash LC (silica gel, gradienthex:EtOAc 2:1 to 1:1) to give SAPL3 (7.98 g, 89% yield) as a whitesolid. Rf=0.18 (hex/EtOAc 1:1).

¹H NMR (300 MHz, CDCl₃) δ 0.79-1.00 (m, 24H), 1.10-2.25 (m, 10H), 1.18(d, J=6.3, 3H), 1.25 (s, 3H), 1.32 (d, J=6.8, 3H), 2.28-2.34 (m, 1H),2.49 (dd, J₁=10.7, J₂=17.0, 1H), 2.54 (s, 3H), 2.83 (s, 3H), 2.95 (m,1H), 3.02-3.24 (m, 2H), 3.31-3.40 (dd, J₁=3.9, J₂=14.1, 1H), 3.53-3.64(m, 2H), 3.65-3.75 (m, 1H), 3.78 (s, 3H), 3.92-4.20 (m, 3H), 4.58 (t,J=4.8, 1H), 4.75-4.85 (m, 3H), 5.00 (m, 1H), 5.12-5.26 (m, 3H), 6.84 (d,J=8.3, 2H), 6.86 (bs, 1H), 7.07 (d, J=8.3, 2H), 7.21-7.44 (min 6H), 7.92(d, J=8.3, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.57, 14.05, 15.18, 16.73, 18.52, 20.83,22.62, 22.96, 23.66, 24.55, 24.81, 25.03, 25.28, 26.90, 27.86, 28.95,31.28, 31.81, 33.91, 34.02, 38.51, 38.61, 47.03, 49.61, 55.21, 55.38,55.50, 57.28, 66.10, 67.65, 67.93, 70.47, 81.44, 114.09, 127.83, 128.46,129.74, 130.29, 158.60, 168.36, 169.59, 170.28, 171.20, 172.22, 204.80.

ESI-MS Calcd for C₅₇H₈₄N₆O₁₄ 1076.60. Found m/z 1077.6 (M+H)⁺.

Example 51 Synthesis of [Aip]³ Z-Didemnin A (SNPL3)

Following the procedure described for the synthesis of SAPL3, startingfrom SNPL4 (35 mg, 41 □mol) and H1 (17 mg, 61 □mol), the title compound(36 mg, 81%) was obtained as a white solid after purification by flashLC (silica gel, gradient hex-EtOAc from 1:4 to EtOAc neat). Rf=0.30(EtOAc).

¹H NMR (300 MHz, CDCl₃) δ 0.74 (d, J=6.3, 3H), 0.80-1.00 (m, 21H),1.10-2.25 (m, 10H), 1.21 (d, J=5.8, 3H), 1.24 (s, 3H), 1.34 (d, J=6.3,3H), 2.61 (s, 3H), 2.86 (s, 3H), 3.12-3.25 (dd, J₁=11.2, J₂=14.1, 1H),3.27-3.36 (dd, J₁=4.3, J₂=14.1, 1H), 3.52-3.63 (m, 3H), 3.69-3.81 (m,2H), 3.80 (s, 3H), 4.09 (m, 3H), 4.47-4.63 (m, 3H), 4.76-4.92 (m, 2H),5.00 (m, 1H), 5.08 (m, 1H), 5.18 (s, 2H), 6.85 (d, J=8.3, 2H), 6.97 (d,J=6.97, 1H), 7.07 (d, J=8.3, 2H), 7.35 (bs, 5H), 7.48 (d, J=8.3, 1H),7.67 (d, J=8.3, 1H), 7.87 (d, J=10.2, 1H).

ESI-MS Calcd for C₅₇H₈₅N₇O₁₃: 1075.62. Found m/z: 1076.6 (M+H)⁺.

Example 52 Synthesis of [Hiv]³ Z-Didemnin A (SHPL3)

Following the procedure described for the synthesis of SAPL3, startingfrom SHPL4 (116 mg, 0.15 mmol) and H1 (63 mg, 0.23 mmol), the titlecompound (86 mg, 52%) was obtained as a white solid after purificationby flash LC (silica gel, gradient hex-EtOAc from 1:1 to 1:2). Rf=0.27(hex-EtOAc 1:2).

¹H NMR (300 MHz, CDCl₃) δ 0.80-1.08 (m, 24H), 1.18 (m, 3H), 1.21 (m,4H), 1.58 (m, 2H), 1.74 (m, 1H), 1.80-2.42 (m, 6H), 2.56 (s, 3H), 2.80(s, 3H), 2.88 (m, 1H), 3.15 (m, 1H), 3.32 (m, 1H) 3.60 (m, 3H), 3.78 (s,3H), 3.83 (m, 1H), 3.98 (m, 1H), 4.42 (m, 1H), 4.58 (m, 1H), 4.75 (m,1H), 4.84 (m, 1H), 4.92 (d, J=3.8, 1H), 5.00 (m, 1H), 5.20 (m, 2H), 6.65(d, J=6.3, 1H), 6.84 (d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.34 (m, 5H),7.50 (d, J=6.7, 1H), 7.75 (d, J=7.2, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.58, 14.21, 15.39, 17.64, 18.63, 20.74,21.88, 22.84, 23.48, 24.22, 24.75, 27.05, 27.84, 29.34, 29.99, 33.42,33.86, 35.81, 38.52, 39.37, 46.63, 48.14, 55.13, 55.47, 55.53, 55.87,56.92, 65.68, 67.72, 68.60, 70.61, 79.09, 113.95, 127.71, 127.86,128.35, 129.63, 130.22, 158.48, 168.46, 169.36, 169.84, 170.29, 170.93,171.00, 173.73.

ESI-MS: Calcd for C₅₄H₈₀N₆O₁₃ 1020.58. Found m/z 1021.5 (M+H)⁺.

Example 53 Synthesis of [Val]³ Z-Didemnin A (SVPL3)

Following the procedure described for the synthesis of SAPL3, startingfrom SVPL4 (20 mg, 25 □mol) and SAPLH1 (11 mg, 37.5 □mol), the titlecompound (19 mg, 72%) was obtained as a white solid after purificationby flash LC (silica gel, gradient hex-EtOAc from 1:1 to 1:5). Rf=0.44(EtOAc).

¹H NMR (300 MHz, CDCl₃): δ 0.85-1.06 (m, 21H), 1.10-1.4 (m, 5H), 1.16(d, J=6.6, 3H), 1.50-1.63 (m, 6H), 1.72-1.87 (m, 2H), 1.88-2.40 (m, 6H),2.58 (s, 3H), 2.86 (s, 3H), 3.17 (dd, J₁=10.5, J₂=14.2, 1H), 3.36 (dd,J₁=3.9, J₂=14.2, 1H), 3.43 (bs, 1H), 3.51-3.72 (m, 4H), 3.79 (s, 3H),3.98-4.16 (m, 1H), 4.40-4.47 (m, 2H), 4.58 (dd, J₁=5.7, J₂=7.8, 1H),4.67-4.85 (m, 2H), 4.80-5.09 (m, 1H), 5.16 (d, J=12.4, 1H), 5.24 (d,J=12.4, 1H), 6.84 (d, J=8.4, 1H), 6.90-6.94 (bs, 1H), 7.09 (d, J=8.4,1H), 7.28-7.50 (m, 6H).

¹³C NMR (75 MHz, CDCl₃) δ 11.99, 14.37, 15.70, 18.55, 19.82, 21.40,21.99, 22.81, 23.17, 23.98, 24.62, 25.00, 25.24, 27.28, 28.26, 29.93,30.23, 33.58, 34.10, 36.16, 38.78, 41.98, 47.10, 48.79, 54.73, 55.45,56.62, 57.40, 59.51, 66.02, 68.18, 70.37, 71.03, 114.30, 128.00, 128.27,128.72, 129.86, 130.56, 136.49, 158.28, 158.85, 168.75, 169.27, 170.40,170.75, 171.04, 173.91, 175.03.

ESI-MS Calcd for C₅₄H₈₁N₇O₁₂: 1019.59. Found (m/z): 1020.5 (M+H)⁺.

Example 54 Synthesis of Didemnin A (SAPL2)

To a solution of SAPL3 (6.59 g, 6.1 mmol) in THF (free of stabilizer,262 mL) degassed and purged with argon, Pd(OH)₂—C (20%, 3.29 g, 50% w/w)was added. The mixture was stirred under H2 (1 atm) for 20 h, thenfiltered over a 0.450m teflon filter and concentrated under reducedpressure to give a white solid. CHCl₃ (2×25 ml) was added, and themixture was concentrated again under reduced pressure to give SAPL2(5.51 g, 96%) as a white solid. Rf=0.22 (CHCl₃:tBuOH 90:10).

¹H NMR (500 MHz, CDCl₃) δ 0.82-0.92 (m, 24H), 1.11-1.19 (m, 1H), 1.22(d, J=6.9, 3H), 1.32 (d, J=6.8, 3H), 1.30-1.35 (m, 1H), 1.35-1.63 (m,6H), 1.71-1.81 (m, 2H), 1.93-2.07 (m, 1H), 2.07-2.18 (m, 2H), 2.28-2.34(m, 1H), 2.49-2.52 (dd, J₁=11, J₂=10.5, 1H), 2.54 (s, 3H), 2.72 (bs,3H), 2.79 (bs, 3H), 2.86-2.94 (bs, 1H), 2.72-2.79 (bd, J=10.5, 1H),3.15-3.18 (dd, J₁=14.5, J₂=10.5, 1H), 3.33-3.36 (dd, J₁=14.5, J₂=4.5,1H), 3.54-3.57 (dd, J₁=10.5, J₂=4.5, 1H), 3.56-3.61 (m, 1H), 3.78 (s,3H), 3.96-4.00 (m, 1H), 4.03-4.08 (m, 1H), 4.11-4.80 (bs, 1H), 4.56-4.62(m, 1H), 4.68-4.81 (m, 3H), 4.99-5.01 (q, J=3.5, 1H), 5.16 (bs, 1H),6.83 (d, J=8.5, 2H), 6.95 (bs, 1H), 7.07 (d, J=8.5, 2H), 7.21-7.25 (bs,1H), 7.95 (bs, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.55, 14.95, 15.26, 16.82, 18.56, 20.89,22.00, 23.08, 23.76, 24.58, 24.85, 25.10, 27.12, 29.35, 29.35, 29.65,29.69, 31.36, 33.96, 34.14, 38.51, 38.64, 40.14, 55.38, 55.56, 57.31,66.17, 67.85, 70.58, 80.96, 80.98, 81.57, 114.12, 130.33, 158.63,168.41, 169.33, 169.70, 170.38, 171.24, 172.28, 172.28, 172.93, 204.83.

m/z (FAB) 944.2 [(M+H)⁺, 100].

Example 55 Synthesis of [Aip]³-Didemnin A (SNPL2)

To a solution of SNPL3 (33 mg, 35 □mol) in a mixture of IPA/H₂O (2 ml: 1ml) degassed and purged with argon, Pd(OH)₂—C (20%, 20 mg, 60% w/w) wasadded. The mixture was stirred under H₂ (1 atm) for 20 h, then filteredover a 0.45 mm teflon filter and concentrated under reduced pressure togive a white solid. IPA (2×5 mL) was added, and the solution wasconcentrated again under reduced pressure to give SNPL2 (32 mg, 97%) asa white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.78 (d, J=6.8, 3H), 0.82-0.92 (m, 21H),1.11-2.58 (m, 21H), 2.41 (s, 3H), 2.62 (s, 3H), 2.75-3.00 (m, 4H),3.15-3.18 (dd, J₁=10.7, J₂=14.7, 1H), 3.33-3.36 (dd, J₁=14.5, J₂=4.2,1H), 3.51-3.78 (m, 3H), 3.78 (s, 3H), 3.92 (m, 1H), 4.01-4.20 (m, 2H),4.50 (t, J=4.8, 1H), 4.59 (t, J=6.3, 1H), 4.75-4.91 (m, 2H), 5.05 (m,1H), 6.84 (d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.70 (d, J=5.8, 1H), 7.78(d, J=9.7, 1H), 7.89 (d, J=6.3, 1H), 8.14 (d, J=7.8, 1H).

ESI-MS Calcd for C₄₉H₇₉N₇O₁₁ 941.58. Found m/z 942.7 (M+H)⁺.

Example 56 Synthesis of [Hiv]³-Didemnin A (SHPL2)

Following the procedure described for the synthesis of SAPL2, startingfrom SHPL3 (86 mg, 0.08 mmol), the title compound (73 mg, 97%) wasobtained as a white solid. Rf=0.36 (CHCl₃/MeOH 95:5).

¹HNMR (300 MHz, CDCl₃) 80.82-1.02 (m, 24H), 1.12-2.42 (m, 16H), 2.54 (s,3H), 2.64 (s, 3H), 2.95 (m, 1H), 3.15 (m, 1H), 3.35 (m, 1H), 3.52-3.90(m, 5H), 3.78 (s, 3H), 4.04 (m, 1H), 4.38 (m, 1H), 4.48 (m, 1H), 4.57(m, 1H), 4.88 (m, 1H), 4.91 (d, J=5.3, 1H), 5.22 (m, 1H), 6.84 (d,J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.54 (d, J=9.2, 1H), 7.60 (d, J=9.4,1H), 8.68 (d, J=6.2, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.78, 14.07, 16.13, 17.90, 18.56, 20.98,21.74, 22.94, 23.60, 24.44, 24.78, 25.06, 27.17, 27.92, 30.09, 33.34,33.89, 38.71, 40.30, 46.86, 48.22, 54.99, 55.23, 56.97, 57.21, 65.81,68.53, 70.37, 79.47, 114.03, 129.76, 130.29, 158.57, 168.18, 169.40,169.86, 170.20, 170.92, 174.16.

ESI-MS Calcd for C₄₆H₇₄N₆O₁₁ 886.54. Found m/z 887.2 (M+H)⁺.

Example 57 Synthesis of [Val]³-Didemnin A (SVPL2)

Following the procedure described for the synthesis of SAPL2, startingfrom SVPL3 (19 mg, 18 □mol), the title compound (16 mg, 97%) wasobtained as a white solid. Rf=0.36 (CHCl₃/MeOH 95:5).

¹H NMR (300 MHz, CDCl₃): δ−0.82-1.02 (m, 21H), 1.85-1.32 (m, 9H),1.42-1.83 (m, 7H), 1.87-2.23 (m, 8H), 2.34 (bs, 3H), 2.60 (b s, 1H),2.85 (dd, J₁=5.7, J₂=7.8, 1H), 3.16 (dd, J₁=10.8, J₂=14.2, 1H), 3.37(dd, J₁=4.2, J₂=14.2, 1H), 3.48 (bs, 1H), 3.57-3.68 (m, 4H), 3.79 (s,3H), 3.95-4.15 (m, 2H), 4.45-4.52 (m, 2H), 4.61 (dd, J₁=4.8, J₂=7.8,1H), 4.77 (t, J=9.9, 1H), 5.02-5.15 (m, 1H), 6.84 (d, J=8.4, 1H), 7.09(d, J=8.4, 1H), 7.59-7.62 (m, 2H), 7.78-7.81 (m, 2H).

¹³C NMR (75 MHz, CDCl₃) δ311.98, 14.46, 16.14, 18.50, 19.83, 21.50,22.55, 22.81, 22.95, 23.46, 23.98, 27.33, 28.27, 28.33, 29.94, 33.71,34.16, 38.94, 41.93, 42.11, 45.72, 47.18, 48.94, 53.64, 55.03, 55.50,56.72, 57.55, 59.22, 59.56, 66.16, 70.71, 114.36, 118.53, 120.85,130.02, 130.58, 168.71, 169.74, 170.23, 171.18, 175.10.

ESI-MS Calcd for C₄₆H₇₅N₇O₁₀: 885.56. Found (m/z): 856.5 (M+H)⁺.

Example 58 Synthesis of Pyr-Pro-OBn (F2)

To a solution of H-Pro-OBn.HCl (10.0 g, 41.3 mmol) in anh DMF (50 mL) at0° C. under Ar, NMM (4.55 mL, 43.8 mmol) was added dropwise by syringewhile maintaining the temperature at 0° C. HOBt (18.98 g, 124 mmol) wasthen added in portions. After 15 min, pyruvic acid (8.61 g, 97.79 mmol)dissolved in anh DCM (10 mL) was added dropwise by syringe whilemaintaining the temperature below 3° C. Finally, DCC (22.17 g, 107.46mmol) dissolved in DCM (80 mL) was added dropwise with a compensatedfunnel. The mixture was allowed to reach room temperature (2 h) andstirred for another 12 h. The reaction mixture was filtered to removethe precipitate and the solution was concentrated under reducedpressure. The residue was dissolved in EtOAc (500 mL) and washedsuccessively with aq. KHSO₄ (100 mL, 10%), aq. NaHCO₃ (400 mL, sat.),brine (400 mL), dried (Na₂SO₄) and filtered. The solvent was eliminatedunder reduced pressure and the residue was chased with ACN (100 mL),cooled at −30° C. for 2 h and filtered to removed the excess ofN,N-dicyclohexylurea. The resulting brown oil (15.82 g) was purified byflash LC (silica gel, grad. hex to hex:EtOAc 2:1) to afford F2 (9.06 g,66% yield) as a white solid. Rf=0.25 (hex:EtOAc 2:1).

IR (film, DCM) ν 3035, 2956, 2884, 1744, 1717, 1645, 1499, 1443, 1383,1352, 1273, 1175, 1092 cm⁻¹.

¹H NMR (200 MHz, CDCl₃) δ 1.75-2.40 (m, 4H), 2.37 (s, 3H), 2.44 (s, 3H),3.45-3.82 (m, 2H), 4.52-4.61 (m, 1H), 4.88-4.97 (m, 1H), 5.14-5.15 (m,2H), 5.17-5.20 (m, 2H), 7.34 (bs, 5H).

¹³C NMR (50 MHz, CDCl₃) δ 22.11, 25.22, 26.5, 27.10, 28.53, 31.48,47.53, 44.81, 59.76, 67.02, 67.31, 128.11, 128.64, 135.24, 170.1, 170.2,198.0.

m/z (CI) 293 [(M+NH₄)⁺, 100]. Anal. Calcd for Cl₅H₁₇NO₄: C, 65.44; H,6.22; N, 5.08. Found: C, 65.04; H, 6.01; N, 5.11.

Example 59 Synthesis of Pyr-Pro-OH (F1)

A solution of F2 (8.63 g, 31.34 mmol) and palladium on activatedcharcoal (10%, 86 mg, 10% w/w) in degassed MeOH (125 mL) was placed in ahigh pressure Parr reactor and purged with nitrogen gas (2×30 psi). Thereaction mixture was sealed under hydrogen (30 psi) and stirred at 23°C. for 4.5 h. The mixture was filtered through a 0.45 □m teflon filterand concentrated at reduced pressure. The residue (6.29 g) was coated inSiO₂ and loaded onto the top of a column (LC 5.5×10.0 cm) and elutedwith hex:EtOAc 1:2 to yield F1 (4.64 g, 80%) as a white solid. Rf=0.22(hex:EtOAc 1:1). [α]_(D) ²⁰−92.4 (c 0.12, CHCl₃). M.p. 67-69° C.

IR (film, DCM) ν 3450-3000, 2961-2870, 1719, 1643, 1615, 1452, 1354,1205, 1175, 1094, 1018 cm⁻¹.

¹H NMR (200 MHz, CDCl₃) δ 1.85-2.45 (m, 4H), 2.43 and 2.47 (s, 3H),3.42-3.85 (m, 2H), 4.52-4.61 (m, 1H), 4.88-4.97 (m, 1H), 7.21-7.40 (bs,1H).

¹³C NMR (75 MHz, CDCl₃) δ 22.03, 25.23, 26.48, 27.00, 28.23, 31.44,47.57, 48.37, 59.61, 59.39, 162.47, 162.52, 175.04, 176.29, 197.18.

m/z (CI) 220 [(M+N₂H₇)⁺, 15], 203 [(M+NH₄)⁺, 100], 186 [(M+H)⁺, 16].Anal. Calcd for C₈H₁₁NO₄: C, 51.88; H, 5.99; N, 7.56. Found: C, 52.13;H, 5.85; N, 7.65

Example 60 Synthesis of Aplidine (SAPL1)

To a cold (3° C.) solution of F1 (4.908 g, 26.5 mmol) in anh. DCM (40mL), was added, under nitrogen, a solution of DIPCDI (1.806 mg, 14.3mmol) in DCM (10 ml) and the solution was stirred at 3° C. for 60 min.Then, a solution of SAPL2 (5.0 g, 5.3 mmol) in DCM (50 ml) wastransferred via cannula to the previous solution under nitrogenpressure. After 90 h (4 days) at this temperature, aq HCl (50 ml, 0.1N)was added, and the reaction mixture was stirred for 15 min. Then, theorganic layer was decanted and partitioned between aq. KHSO₄ (50 mL,5%), aq. NaHCO₃ (50 mL, 5%) and brine (25 mL). The organic phase wasdried (Na₂SO₄), filtered and concentrated under reduced pressure. Theresulting pale yellow solid was purified by flash LC (Lichroprep RP-18,40-63 □m, gradient MeOH:H₂O:TFA from 70:30:0.1 to 90:10:0.1) to yieldSAPL1 (5.4 g, 93%) (mixture of rotamers). Rf=0.40 and 0.28 (DCM:AcOEt,2:3), 0.52 and 0.45 (CHCl₃-MeOH, 9.5:0.5); [α]_(D)−95.9 (c 1.8, CHCl₃).

¹H NMR (500 MHz, CDCl₃) δ 0.84-0.93 (m, 24H), 1.16-1.70 (m, 9H),1.72-1.81 (m, 1H), 1.81-1.90 (m, 1H), 1.90-2.24 (m, 6H), 2.30-2.39 (m,1H), 2.49 (s, 3H) 2.51 (s, 3H), 2.55 (s, 3H), 2.52-2.64 (m, 1H), 2.85(bs, 1H), 2.94 (bs, 1H), 3.09 (s, 3H), 3.13 (s, 3H), 3.15-3.18 (m, 1H),3.21-3.26 (dd, J₁=15.8, J₂=6.1, 1H), 3.32-3.36 (dd, J₁=14.5, J₂=4.1,1H), 3.54-3.60 (m, 1H), 3.66-3.72 (m, 1H), 3.78 (s, 3H), 3.80-3.87 (m,1H), 3.96-3.99 (m, 1H), 4.03-4.11 (m, 2H), 4.15-4.23 (2q, J=7.5, 1H),4.55-4.57 (2d, J₁=5.5, J₂=2.2, 1H), 4.59-4.62 (t, 1H), 4.56-4.64 (dd,J₁=6.5, J₂=2.5, 1H), 4.68-4.71 (t, 1H), 4.76-4.81 (t, 1H), 5.10-5.18 (m,1H), 5.17 (d, J=3.5, 1H), 5.18 (d, J=3.5, 1H), 5.27-5.31 (m, 2H), 6.82(d, J=8.5, 2H), 6.83 (d, J=8.5, 2H), 7.05 (d, J=8.5, 2H), 7.06 (d,J=8.5, 2H), 7.02 (d, J=7.1, 1H), 7.16 (d, J=9.5, 1H), 7.17 (d, J=9.5,1H), 7.59 (d, J=5.5, 1H), 7.77 (d, J=9.5, 1H), 7.83 (d, J=9.4, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.63, 11.68, 14.11, 14.70, 15.26, 15.30,16.00, 16.20, 16.88, 16.93, 18.62, 18.85, 20.89, 20.94, 21.62, 21.36,23.44, 23.57, 23.84, 23.93, 24.66, 24.77, 24.85, 25.02, 26.22, 26.34,27.09, 27.6, 27.06, 27.30, 27.95, 27.99, 29.33, 29.69, 31.31-31.37,33.97, 34.06, 36.02, 36.45, 38.68, 38.76, 41.01, 41.15, 47.00, 48.42,48.48, 48.86, 49.20, 49.51, 54.65, 54.75, 55.26, 55.58, 55.61, 57.14,57.27, 57.47, 57.79, 66.24, 67.80, 67.99, 70.34, 70.67, 81.0, 81.52,114.10, 130.31, 156.0, 158.65, 161.1, 161.60, 168.20, 169.53, 169.59,170.45, 171.25, 171.80, 171.95, 172.26, 172.33, 197.5, 204.80, 204.85.

m/z (FAB) 1132.6 [(M+Na)⁺, 42], 1110.8 [(M+H)⁺, 100].

Example 61 Synthesis of [Aip]³-Aplidine (SNPL1)

Following the procedure described for the synthesis of SAPL1, startingfrom SNPL2 (10 mg, 10.6 □mol) and F1 (10 mg, 54 □mol). The titlecompound (8 mg, 68%) was obtained as a white solid after purification byHPLC (HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min,250×21 mm, at 270 nm, t_(R)=10.5 and 12.0 min).

¹H NMR (300 MHz, CDCl₃) δ 0.80-1.03 (m, 24H), 1.11-1.70 (m, 9H),1.72-1.81 (m, 1H), 1.81-1.90 (m, 1H), 1.90-2.24 (m, 6H), 2.30-2.39 (m,1H), 2.53 (s, 3H) 2.55 (s, 3H), 2.65 (s, 3H), 2.52-2.66 (m, 2H), 2.94(m, 1H), 3.07 (s, 3H), 3.11 (s, 3H), 3.15-3.18 (m, 1H), 3.22-3.31 (dd,J₁=4.3, J₂=15.1, 1H), 3.54-3.60 (m, 2H), 3.67-3.92 (m, 2H), 3.80 (s,3H), 3.98 (m, 1H), 4.13-4.29 (m, 3H), 4.45-4.75 (m, 4H), 4.81 (t, J=9.7,1H), 5.09 (m, 1H), 5.18 (m, 1H), 5.26-5.44 (m, 3H), 6.84 (d, J=8.3, 2H),7.07 (d, J=8.3, 2H), 7.30 (d, J=8.3, 1H), 7.36 (d, J=8.3, 1H), 7.68 (d,J=9.7, 1H), 7.87 (d, J=4.3, 1H), 8.09 (d, J=9.7, 1H), 8.28 (d, J=10.2,1H).

ESI-MS Calcd for C₅₇H₈₈N₈O₁₄: 1108.64. Found (m/z): 1110.3 (M+H)⁺.

Example 62 Synthesis of [Hiv]³-Aplidine (SHPL1)

Following the procedure described for the synthesis of SAPL1, startingfrom SHPL2 (72 mg, 0.081 mmol) and F1 (75 mg, 0.405 mmol). The titlecompound (68 mg, 79%) was obtained as a white solid after purificationby flash LC (Lichroprep RPC18, gradient ACN/H₂O/TFA from 70:30:0.5 to90:10:0.5). Rf=0.49 (ACN/H₂O/TFA 90:10:1).

¹H NMR (300 MHz, CDCl₃) δ 0.80-1.10 (m, 24H), 1.12-1.50 (m, 18H),1.50-2.30 (m, 6H), 2.42 (m, 1H), 2.53 (s, 3H) 2.55 (s, 3H), 2.57 (s,3H), 2.96-3.40 (m, 3H), 3.05 (s, 3H), 3.10 (s, 3H), 3.63 (m, 5H), 3.78(s, 3H), 3.90 (m, 1H), 4.01 (m, 1H) 4.30 (m, 1H), 4.63 (m, 1H), 4.69 (m,1H), 4.86 (m, 1H), 5.02 (d, J=4.8, 1H), 5.09 (m 1H), 5.20 (m, 1H), 5.30(m, 1H), 6.83 (d, J=8.3, 2H), 6.89 (d, J=6.3, 1H), 7.07 (d, J=8.3, 2H),7.29 (d, J=9.7, 1H), 7.34 (m, 2H), 7.43 (d, J=5.3, 1H), 7.74 (d, J=9.7,1H), 7.80 (d, J=10.2, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 12.06, 14.22, 14.30, 16.49, 16.76, 17.84,19.17, 21.08, 21.41, 21.54, 22.54, 23.79, 23.94, 24.05, 24.16, 24.82,24.96, 25.05, 25.98, 26.45, 27.37, 27.57, 28.21, 28.59, 30.31, 30.83,31.56, 31.62, 33.74, 34.24, 36.02, 36.25, 38.91, 38.96, 39.46, 39.86,46.92, 48.44, 48.71, 49.06, 54.95, 55.49, 57.16, 57.68, 58.23, 59.13,66.16, 66.28, 69.10, 70.83, 71.14, 79.12, 114.31, 129.96, 130.12,130.59, 158.86, 168.69, 168.81, 169.75, 169.82, 170.18, 170.45, 170.52,170.69, 170.84, 171.21, 171.28, 172.47, 173.17, 174.66, 174.82, 197.63,201.38.

ESI-MS Calcd for C₅₄H₈₃N₇O₁₄: 1053.60. Found (m/z): 1054.9 (M+H)⁺.

Example 63 Synthesis of [Val]³-Aplidine (SVPL1)

Following the procedure described for the synthesis of SAPL1, startingfrom SVPL2 (10 mg, 11 □mol) and F1 (10.5 mg, 57 □mol). The titlecompound (8 mg, 69%) was obtained as a white solid after purification byHPLC (HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min,250×21 mm, at 270 nm, t_(R)=10.9 and 12.3 min).

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.02 (m, 24H), 1.13-1.38 (m, 9H), 1.55(m, 2H), 1.67-1.81 (m, 4H), 1.95-2.02 (m, 3H), 2.10-2.17 (m, 2H),2.26-2.39 (m, 2H), 2.56 (s, 3H), 2.57 (s, 3H), 2.58 (s, 3H), 2.74-2.92(m, 1H), 3.10 (s, 3H), 3.15 (s, 3H), 3.20 (m, 1H), 3.36 (dd, J₁=4.4,J₂=14.2, 1H), 3.49-3.72 (m, 5H), 3.79 (s, 3H), 3.97-4.13 (m, 2H), 4.38(dd, J₁=4.6, J₂=14.2, 1H), 4.49 (m, 1H), 4.60 (m, 1H), 4.68-4.81 (m,2H), 5.11 (m, 1H), 5.26-5.30 (m, 1H), 5.33-5.40 (m, 1H), 6.84 (d, J=7.8,2H), 7.08 (d, J=8.3, 2H), 7.36-7.52 (m, 2H), 7.48 (d, J=9.6, 1H), 7.61(d, J=6.8, 1H).

ESI-MS Calcd for C₅₄H₈₄N₈O₃ 1052.62. Found (m/z): 1053.6 (M+H)⁺.

Example 64 Synthesis of [Hiv]³-[isobutyryl]⁸-didemnin A (8ISHPL1)

To a solution of SHPL2 (10 mg, 11.2 □mol) in DCM (200 □l) at 0° C. underAr, was added DIPEA (3 □l, 16.8 □mol) and isobutyryl chloride (1.4 □l,13.4 μmol). After 3 h at 22° C., DCM (3 ml) was added and the mixturewas washed successively with aq. HCl (2 ml, 0.1N), aq. NaHCO₃ (2 ml,sat.) and brine (2 ml), dried (Na₂SO₄), filtered and concentrated invacuo. Purification of the residue by HPLC (HyperPrep PEP 100 C18,isocratic ACN/H₂O 85:15 (flow: 7 ml/min, 250×21 mm, at 270 nm, t_(R)=19min) afforded the title compound (10 mg, 94%) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.10 (24H, m), 1.13-1.65 (18H, m),1.72-2.58 (6H, m), 2.56 (s, 3H), 2.89 (s, 3H), 2.92 (m, 2H), 3.13 (m,2H), 3.36 (dd, J₁=4.6, J₂=15.6, 1H), 3.54-3.73 (m, 3H), 3.78 (s, 3H),3.91 (m, 2H), 4.40 (m, 1H), 4.60 (m, 1H), 4.89 (m, 1H), 4.99 (d, J=5.3,1H), 5.03 (m, 1H), 5.20 (m, 1H), 6.73 (d, J=9.3, 1H), 6.84 (d, J=9.6,2H), 7.08 (d, J=9.6, 2H), 7.55 (d, J=8.6, 1H), 7.82 (d, J=11, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.78, 14.63, 15.69, 17.92, 19.03, 19.31,19.66, 21.03, 22.29, 23.19, 23.85, 24.71, 25.14, 27.27, 28.22, 30.39,30.51, 31.15, 33.80, 34.25, 35.61, 38.85, 39.63, 46.98, 48.51, 53.29,53.65, 55.49, 56.01, 56.35, 57.26, 66.07, 69.07, 70.94, 79.33, 114.31,130.08, 130.60, 158.84, 168.79, 169.75, 170.34, 170.68, 171.36, 171.73,174.02, 179.84.

ESI-MS Calcd for C₅₀H₈₀N₆O₁₂, 956.58. Found (m/z): 957.5 (M+H)⁺.

Example 65 Synthesis of [Val]³-[Isobutyryl]⁸-didemnin A (8ISVPL1)

Following the procedure described for the synthesis of 8ISHPL1, startingfrom SVPL2 (20 mg, 22.6 □mol). The title compound (19 mg, 88%) wasobtained after purification by HPLC (HyperPrep PEP 100 C18, isocraticACN/H₂O 85:15, flow: 7 ml/min, 250×21 mm, at 270 mm, t_(R)=19 min).

¹H NMR (300 MHz, CDCl₃): δ 0.81-1.02 (m, 24H), 1.14-1-38 (m, 5H), 1.15(d, J=6.6, 3H), 1.19 (d, J=6.6, 3H), 1.38-1.80 (m, 7H), 1.80-2.40 (m,6H), 2.57 (s, 3H), 2.58-2.64 (m, 1H), 2.85-2.92 (m, 1H), 2.93 (s, 3H),3.16 (dd, J₁=10.5, J₂=14.4, 1H), 3.36 (dd, J₁=4.5, J₂=14.4, 1H), 3.39(bs, 1H), 3.56 (dd, 1H, J=4.5, 10.8), 3.59-3.72 (m, 3H), 3.78 (s, 3H),4.01 (td, J₁=3.3, J₂=10.2, 1H), 4.39-4.47 (m, 2H), 4.58 (dd, J₁=5.7,J₂=7.5, 1H), 4.79 (t, J=9.9, 1H), 5.03-5.14 (m, 2H), 6.84 (d, J=8.4,2H), 7.04 (d, J=7.8, 1H), 7.08 (d, J=8.4, 1H), 7.36 (d, J=9.0, 1H), 7.45(d, J=10.2, 1H), 7.51 (d, J=10.2, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 180.24, 175.10, 173.18, 171.14, 170.72,170.42, 169.38, 168.73, 158.87, 130.60, 130.02, 114.33, 71.31, 70.50,66.11, 59.47, 57.42, 56.56, 55.51, 54.93, 53.84, 48.83, 47.13, 41.94,38.94, 35.67, 34.17, 33.63, 31.32, 31.10, 29.96, 28.32, 27.25, 25.30,25.05, 24.82, 24.01, 23.22, 21.42, 19.88, 19.72, 11.25, 18.51, 15.60,14.32, 11.96.

ESI-MS Calcd for C₅₀H₈₁N₇O₁₁: 955.60. Found 956.8 (M+H)⁺.

Example 66 Synthesis of [Hiv]³-[Butyryl]⁸-didemnin A (8BSHPL1)

Following the procedure described for the synthesis of 81SHPL1, startingfrom SHPL2 (10 mg, 11.2 □mol). The title compound (9 mg, 84%) wasobtained as a white solid after purification by HPLC (HyperPrep PEP 100C18, isocratic ACN/H₂O 85:15, flow: 7 ml/min, 250×21 mm, at 270 nm,t_(R)=18.6 min).

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.10 (m, 24H), 1.11-1.72 (m, 18H),1.75-2.51 (m, 6H), 2.56 (s, 3H), 2.84 (s, 3H), 2.92 (m, 2H), 3.15 (m,2H), 3.35 (dd, J₁=5.0, J₂=15.6, 1H), 3.54-3.77 (m, 3H), 3.79 (s, 3H),3.91 (m, 2H), 4.40 (m, 1H), 4.60 (m, 1H), 4.89 (m, 1H), 4.98 (d, J₁=6.0,1H), 5.04 (m, 1H), 5.20 (m, 1H), 6.78 (d, J₁=9.6, 1H), 6.84 (d, J₁=9.6,2H), 7.08 (d, J₁=9.6, 2H), 7.54 (d, J₁=9.6, 1H), 7.82 (d, J₁=10.6, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.82, 14.15, 14.55, 15.72, 17.96, 18.67,19.01, 21.05, 22.37, 23.13, 23.84, 24.71, 25.14, 27.30, 28.21, 29.92,30.38, 30.63, 33.78, 34.25, 35.69, 35.97, 38.85, 39.61, 46.98, 48.52,53.27, 55.49, 55.99, 56.25, 57.27, 66.07, 69.07, 70.96, 79.40, 114.31,130.08, 130.59, 158.85, 168.78, 169.73, 170.42, 170.64, 171.36, 171.65,174.07, 175.79.

ESI-MS Calcd for C₅₀H₈₀N₆O₁₂: 956.60. Found (m/z): 957.8 (M+H)⁺.

Example 67 Synthesis of [Hiv]³-[hexanoyl]⁸-didemnin A (8HSHPL1)

Following the procedure described for the synthesis of 81SHPL1, startingfrom SHPL2 (10 mg, 11.2 □mol), the title compound (9 mg, 82%) wasobtained as a white solid, after purification by HPLC (HyperPrep PEP 100C18, isocratic ACN/H₂O 85:15, flow: 7 ml/min, 250×21 mm, at 270 nm,t_(R)=27.8 min).

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.10 (m, 24H), 1.11-1.72 (m, 22H),1.80-2.51 (m, 6H), 2.56 (s, 3H), 2.84 (s, 3H), 2.93 (m, 2H), 3.14 (m,2H), 3.35 (dd, J₁=4.4, J₂=14.1, 1H), 3.54-3.76 (m, 3H), 3.79 (s, 3H),3.91 (m, 2H), 4.41 (m, 1H), 4.60 (m, 1H), 4.88 (m, 1H), 4.98 (d, J₁=5.3,1H), 5.03 (m, 1H), 5.19 (m, 1H), 6.76 (d, J₁=8.7, 1H), 6.84 (d, J₁=8.7,2H), 7.08 (d, J₁=8.7, 2H), 7.51 (d, J₁=8.8, 1H), 7.81 (d, J₁=9.7, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.59, 13.94, 14.34, 15.49, 17.73, 18.77,20.81, 22.14, 22.46, 22.90, 23.60, 24.47, 24.69, 24.90, 27.08, 27.97,30.14, 30.40, 31.54, 33.53, 33.76, 34.02, 35.47, 38.62, 39.40, 46.76,48.28, 53.04, 55.26, 55.78, 55.90, 57.04, 65.84, 68.82, 70.72, 79.17,114.07, 129.84, 130.36, 158.61, 168.54, 169.51, 170.15, 170.41, 171.12,171.42, 173.86, 175.75.

ESI-MS Calcd for C₅₂H₈₄N₆O₁₂: 984.61. Found (m/z); 985.8 (M+H)⁺.

Example 68 Synthesis of Isobutyryl-Pro-OBn

To a solution of H-Pro-OBn.HCl (500 mg, 2.07 mmol) in DCM (10 ml) at 0°C., NMM (680 □l, 6.21 mmol) was added under argon. After 10 min,isobutyryl chloride (240 □l, 2.27 mmol) was added and the reactionmixture was allowed to warm to 20° C. and stirred for 5 h. The mixturewas filtered and the filtrate washed successively with aq. HCl (15 ml,1N), aq NaHCO₃ (10 ml, sat.), and brine (10 ml), then dried (Na₂SO₄),filtered and concentrated under reduced pressure to afford 560 mg (98%)of the title compound. Rf=0.42 (hex:EtOAc 1:1).

¹H-NMR (300 MHz, CDCl₃) δ 1.20-1.40 (2d, 6H), 1.90-2.35 (m, 4H), 2.35(q, 1H), 3.40-3.80 (m, 2H), 4.30 (m, 1H), 5.20 (m, 2H), 7.40 (m, 5H).

Example 69 Synthesis of Isobutyryl-Pro-OH

To a solution of Isobutyryl-Pro-OBn (430 mg, 1.56 mmol) in degassed MeOHwas added Pd/C (10%) (43 mg, 10% w/w) and then flushed successively withAr and bubbled with hydrogen. The mixture was stirred under H2 for 14 hand then degassed and filtered. The solution was concentrated and theresidue crystallized with MTBE/hex to give 140 mg (48%) of the titlecompound as a white solid.

¹H-NMR (300 MHz, CDCl₃) δ 1.20 (m, 6H), 1.90-2.10 (m, 3H), 2.50 (m, 1H),2.70 (q, 1H), 3.40-3.70 (m, 2H), 4.60 (dd, 1H).

ESI-MS Calcd for C₉H₁₅NO₃: 185.11. Found (m/z): 186.1 (M+H)⁺.

Example 70 Synthesis of [Hiv]³-[Isobutyryl]⁹-aplidine (91SHPL1)

To a solution of Isobutyryl-Pro-OH (10 mg, 54 □mol) in DCM (150 □l) at0° C., was added DIPCDI (5 □l, 32 □mol). Stirring was continued for 60min and then, the mixture was transferred to a flask containing SHPL2(10 mg, 11.2 □mol) in DCM (150 □l). After 4d at 2-4° C. the mixture wasdiluted with DCM (2 ml) and washed successively with aq HCl (1 ml, 0.1N), aq. NaHCO₃ (1 ml, sat.) and brine (1 ml), the organic phase wasdried (Na₂SO₄), filtered and concentrated. The residue was purified byHPLC (HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min,250×21 mm, at 270 nm, t_(R)=13 and 14 min) to afford 9ISHPL1 (9 mg, 72%)as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.10 (m, 24H), 1.12-1.50 (m, 18H),1.52-2.70 (m, 10H), 2.56 (s, 3H), 3.00-3.44 (m, 4H), 3.08 (s, 3H),3.55-3.72 (m, 5H) 3.78 (s, 3H), 4.00 (m, 3H), 4.26 (m, 1H), 4.62 (m,2H), 4.86 (m, 1H), 5.02 (d, J=5.3, 1H), 5.30 (m, 2H), 6.84 (d, J=9.0,2H), 7.07 (d, J=9.0, 2H), 7.29 (d, J=11.0, 1H), 7.80 (d, J=9.0, 1H);7.88 (d, J=11, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 12.04, 14.44, 16.91, 17.94, 18.96, 19.03,19.15, 21.07, 21.59, 23.87, 24.10, 24.89, 25.08, 26.04, 27.54, 28.21,28.87, 30.33, 31.61, 32.64, 33.77, 34.24, 36.19, 39.07, 39.33, 39.81,46.87, 47.52, 48.45, 54.74, 55.49, 56.20, 57.08, 58.50, 66.41, 69.13,71.48, 79.16, 114.27, 130.30, 130.59, 158.80, 168.55, 169.88, 170.86,171.06, 171.22, 173.63, 174.92, 176.19.

ESI-MS Calcd for C₅₅H₈₇N₇O₁₃: 1053.64. Found (m/z): 1054.6 (M+H)⁺.

Example 71 Synthesis of [Val]³-[Isobutyryl]⁹-aplidine (91SVPL1)

Following the procedure described for the synthesis of 9ISHPL1, startingfrom SVPL2 (10 mg, 11.2 □mol), the title compound (9 mg, 77%) wasobtained as a white solid after purification by HPLC (HyperPrep PEP 100C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min, 250×21 mm, at 270 nm,t_(R)=15.3 min).

¹H NMR (300 MHz, CDCl₃): δ0.82-1.01 (m, 24H), 1.14-1.37 (m, 12H),1.148-2.35 (m 8H), 2.55 (s, 3H), 2.57-2.68 (m, 1H), 2.77-2.83 (m, 1H),3.12 (s, 3H), 3.19-3.23 (m, 1H), 3.35-3.41 (m, 2H), 3.52-3.56 (m, 1H),3.58-3.70 (m, 3H), 3.78 (s, 3H), 4.03-4.13 (m, 1H), 4.33-4.35 (m, 1H),4.44-4.48 (m, 1H), 4.55-4.66 (m, 2H), 4.70-4.83 (m, 1H), 5.35-5.39 (m,2H), 6.82 (d, J=8.4, 2H), 7.06 (d, J=8.4, 2H), 7.25 (bs, 2H), 7.37 (d,10.5, 1H), 7.46 (d, J=8.7, 1H), 7.60 (d, J=9.3, 1H), 8.07 (bs, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 12.10, 14.44, 16.84, 18.63, 18.95, 19.92,21.31, 21.61, 23.91, 24.05, 24.86, 25.00, 25.30, 26.06, 27.41, 28.33,28.93, 30.00, 31.76, 32.65, 33.59, 31.16, 36.21, 39.17, 41.71, 42.19,47.08, 47.57, 48.88, 54.37, 54.65, 55.50, 56.15, 57.33, 58.70, 59.33,66.45, 70.92, 71.54, 114.29, 130.28, 130.59, 158.82, 168.41, 170.08,170.52, 170.69, 171.03, 172.65, 173.80, 175.68, 176.42.

ESI-MS Calcd for C₅₅H₈₈N₈O₁₂ 1052.65. Found (m/z): 1053.8 (M+H)⁺.

Example 72 Synthesis of Z-NVa-Pro-OMe

Following the procedure described for the synthesis of F2, from Z-NVa-OH(261 mg, 1.04 mmol), H-Pro-OMe.HCl (156.6 mg, 0.94 mmol), the titlecompound (315 mg, 87%) was obtained as a colourless oil afterpurification by LC-silica (hex-EtOAc, gradient 3:1 to 1:1). R_(f)=0.42(hex-EtOAc 1:1).

¹H NMR (300 MHz, CDCl₃): δ 0.97 (t, J=6.9, 2H), 1.44 (six, J=7.5, 2H),1.53-1.65 (m, 1H), 1.68-1.77 (m, 1H), 1.82-2.09 (m, 3H), 2.17-2.24 (m,1H), 3.43-3.80 (m, 2H), 3.71 (s, 3H), 4.45-4.55 (m, 2H), 5.07 (s, 2H),5.51 (d, J=8.4, 1H), 7.32-7.35 (m, 5H).

Example 73 Synthesis of NVa-Pro-OH

To a solution of Z-NVa-Pro-OMe (36 mg, 99 □mol) in a mixture of THF andMeOH (130 □l:130 □l) at 0° C., aq LiOH (130 □l, 15% w/w) was added.After 6 h of stirring the reaction mixture was partitioned between H₂O(3 ml) and diethyl ether (3×2 ml). The organic phase was then extractedwith NaHCO₃ (3×2 ml). The combined aqueous phases were neutralized(pH=5) with aq. HCl (0.1 N) and partitioned with Et₂O (3×3 ml). Theorganic phase was dried (Na₂SO₄), filtered and concentrated to give thetitle compound (36 mg, quant) as a colourless oil.

¹H NMR (300 MHz, CDCl₃): δ 0.96 (t, J=6.9, 2H), 1.41 (six, J=7.4, 2H),1.53-1.65-1.77 (m, 2H), 1.82-2.10 (m, 3H), 2.17-2.24 (m, 1H), 3.52-3.81(m, 2H), 4.45-4.58 (m, 2H), 5.07 (bs, 2H), 5.81 (d, J=8.4, 1H),7.30-7.35 (m, 5H), 7.41 (bs, 1H).

ESI-MS Calcd for C₁₈H₂₄N₂O₅: 348.17. Found (m/z): 349.2 (M+H)⁺.

Example 74 Synthesis of [ZNVa-Pro]⁹-aplidine (9NVSAPL2)

Following the procedure described for the synthesis of SAPL1, startingfrom SAPL2 (18 mg, 19 □mol) and Z-NVa-Pro-OH (34 mg, 97 □mol), the titlecompound (16 mg, 66%) was obtained as a white solid after purificationby HPLC (HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min,250×21 mm, at 270 nm, t_(R)=29 min).

¹H NMR (300 MHz, CDCl₃): δ0.84-0.96 (m, 27H), 1.12-1.85 (m, 19H),2.00-2.25 (m, 7H), 2.30-2.40 (m, 1H), 2.54 (s, 3H), 2.62 (dd, J₁=10.5,J₂=17.7, 1H), 2.93 (d, 4.2, 1H), 3.14 (s, 3H), 3.14-3.20 (m, 1H),3.28-3.34 (m, 2H), 3.50-3.67 (m, 4H), 3.77-3.80 (m, 1H), 3.79 (s, 3H),3.82-3.91 (m, 1H), 4.00-4.17 (m, 2H), 4.27 (dd, J₁=6.3, J₂=13.2, 1H),4.43-4.51 (m, 2H), 4.58-4.63 (m, 1H), 4.69-4.75 (m, 1H), 4.77-4.82 (m,1H), 5.07 (d, 1H, J=12.9, 2H), 5.13 (d, J=12.9, 1H), 5.32-5.41 (m, 2H),6.07 (d, J=8.7, 1H), 6.83 (d, J=8.4, 2H), 7.06 (d, J=8.4, 2H), 7.17 (d,J=9.9, 1H), 7.32 (m, 5H), 7.83 (d, J=9.0, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.68, 13.74, 14.56, 15.24, 16.39, 16.85,18.61, 20.94, 21.26, 23.34, 23.70, 24.76, 24.90, 25.02, 26.00, 27.17,27.81, 28.54, 31.29, 31.40, 33.30, 33.85, 33.86, 36.19, 38.62, 38.84,41.31, 46.91, 47.21, 49.38, 49.49, 52.50, 54.96, 55.24, 55.25, 56.50,57.17, 57.96, 62.53, 66.40, 67.93, 70.64, 81.37114.04, 127.89, 127.77,128.32, 129.97, 130.29, 136.84, 156.72, 158.55, 168.48, 169.36, 169.58,170.52, 171.27, 171.71, 172.54, 173.22, 205.08.

ESI-MS Calcd for C₆₇H₁₀₀N₈O₁₆: 127.73. Found (m/z): 1273.7 (M+H)⁺.

Example 75 Synthesis of [Hiv]³-[Z-NVa-Pro]⁹-aplidine (9NVSHPL2)

Following the procedure described for the synthesis of SAPL1, startingfrom SHPL2 (10 mg, 11.2 □mol), and Z-NVa-Pro-OH (20 mg, 56 □mol), thetitle compound (8 mg, 60%) was obtained as a white solid afterpurification by HPLC (HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15(flow: 7 ml/min, 250×21 mm, at 270 nm, t_(R)=26.7 min).

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.10 (m, 24H), 1.12-1.80 (m, 22H),1.82-2.35 (m, 6H), 2.42 (m, 1H), 2.56 (s, 3H), 2.96-3.38 (m, 4H), 3.10(s, 3H), 3.48-3.72 (m, 5H), 3.78 (s, 3H), 3.88 (m, 1H), 4.01 (m, 1H),4.18 (m, 1H), 4.47 (m, 1H), 4.68 (m, 2H), 4.87 (m, 1H), 5.02 (d, J₁=5.3,1H), 5.08 (m, 2H), 5.28 (m, 1H), 5.42 (m, 1H), 6.10 (d, J=8.3, 1H), 6.84(d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.31 (m, 6H), 7.72 (d, J=4.3, 1H),7.78 (d, J=8.7, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 12.06, 13.88, 14.22, 16.85, 17.76, 18.89,19.18, 21.12, 21.47, 23.72, 23.98, 25.08, 26.25, 27.57, 28.15, 28.79,30.32, 31.61, 33.45, 33.72, 34.07, 35.95, 38.92, 39.59, 39.90, 46.87,47.44, 48.46, 52.76, 55.21, 55.49, 56.77, 57.17, 58.37, 66.41, 66.65,69.22, 71.14, 79.07, 114.24, 127.87, 128.00, 128.56, 130.25, 130.55,157.00, 158.79, 168.82, 169.86, 170.36, 170.58, 170.77, 171.30, 171.86,173.28, 174.94.

ESI-MS Calcd for C₆₄H₉₆N₈O₁₅ 1216.7. Found m/z: 1217.5 (M+H)⁺.

Example 76 Synthesis of [NVa-Pro]⁹-aplidine (9NVSAPL1)

A degassed mixture of 9NVSAPL2 (10 mg, 7.8 □mol) and Pd/C (10%, 5 mg) inIPA:H₂O (0.2 ml:0.1 ml), was saturated (and maintained at 1 atm) withhydrogen gas while stirring for 14 h. Then, the mixture was filtered(teflon 0.450m) and concentrated under vacuum to yield the titlecompound (8.8 mg, quant) as a white solid.

¹H NMR (300 MHz, CDCl₃): δ 0.85-0.95 (m, 27H), 1.18-1.51 (m, 18H),1.50-2.45 (m, 7H), 2.59-2.83 (m, 1H), 2.57 (s, 3H), 2.57-2.80 (m, 3H),2.81-2.95 (m, 1H), 3.14 (s, 3H), 3.15-3.40 (m, 3H), 3.52-3.79 (m, 4H),3.79 (s, 3H), 4.45-4.52 (m, 1H), 4.61-4.65 (m, 1H), 4.70-4.85 (m, 2H),5.17 (d, J=3.3, 1H), 5.36-5.39 (m, 2H), 6.84 (d, J=8.1, 2H), 7.86 (d,J=8.7, 2H), 7.19 (d, J=10.2, 1H), 7.82 (d, J=9.0, 1H), 7.80-7.85 (m,1H).

ESI-MS Calcd for C59940N₈O₁₄: 1138.69. Found (m/z): 1139.7 [(M+H)]⁺.

Example 77 Synthesis of [Hiv]³-[NVa-Pro]⁹-aplidine (9NVSHPL1)

Following the procedure described for the synthesis of 9NVSAPL1,starting from 9NVSHPL2 (10 mg, 8.2 □mol), the title compound (8 mg,quant) was obtained as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.10 (m, 30H), 1.12-1.85 (m, 22H),1.92-2.35 (m, 6H), 2.42 (m, 1H), 2.56 (s, 3H), 3.10-3.45 (m, 4H), 3.10(s, 3H), 3.50-3.70 (m, 5H), 3.78 (s, 3H), 3.82 (m, 1H), 4.01 (m, 1H),4.26 (m, 1H), 4.65 (m, 1H), 4.64 (m, 1H), 4.88 (m, 1H), 5.02 (d, J=5.3,1H), 5.32 (m, 2H), 6.84 (d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.38 (d,J=8.7, 1H), 7.60 (d, J=4.3, 1H), 7.80 (d, J=9.3, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 12.03, 14.13, 14.30, 16.80, 17.81, 18.72,19.15, 21.09, 21.55, 23.73, 23.96, 25.07, 26.16, 27.55, 28.17, 28.72,29.91, 30.33, 31.52, 33.80, 34.17, 36.00, 38.94, 39.51, 39.84, 46.88,47.51, 48.45, 55.01, 55.49, 56.91, 57.15, 58.10, 66.37, 69.17, 71.10,79.12, 114.29, 130.21, 130.57, 158.82, 163.66, 168.91, 169.86, 170.38,170.75, 170.82, 171.30, 173.22, 174.79.

ESI-MS Calcd for C₅₆H₉₀N₈O₁₃ 1082.66. Found m/z: 1083.7 (M+H)⁺.

Example 78 Synthesis of [Hiv]³-[L-Lac(OTBDMS)]⁹-aplidine [9LSHPL2(L)]

Following the procedure described for the synthesis of SAPL1, startingfrom SHPL2 (10 mg, 11.2 □mol) and (L)-Lac(OTBDMS)-Pro-OH (17 mg, 56□mol), the title compound (9 mg, 68%) was obtained as a white solidafter purification by HPLC (HyperPrep PEP 100 C18, gradient ACN/H₂O85:15-100:0 in 10 min. (flow: 7 ml/min, 250×21 mm, at 270 nm, t_(R)=30.1min).

¹H NMR (300 MHz, CDCl₃) δ 0.08 (s, 3H), 0.10 (s, 3H), 0.82-1.10 (m,24H), 1.11-1.72 (m, 18H), 1.75-2.51 (m, 6H), 2.41 (m, 1H), 2.56 (s, 3H),3.00-3.40 (m, 5H), 3.11 (s, 3H), 3.53-3.82 (m, 3H), 3.79 (s, 3H), 3.91(m, 2H), 4.02 (m, 1H), 4.27 (m, 1H), 4.50 (m, 1H), 4.63 (m, 2H), 4.87(m, 1H), 5.01 (d, J=4.8, 1H), 5.27 (m, 2H), 6.84 (d, J=8.7, 2H), 7.07(d, J=8.7, 2H), 7.29 (d, J=9.7, 1H), 7.63 (d, J=5.8, 1H), 7.88 (d,J=9.7, 1H).

¹³C NMR (75 MHz, CDCl₃) δ−4.26, −4.12, 12.04, 14.40, 16.91, 17.97,19.14, 20.60, 21.11, 21.66, 23.82, 24.11, 24.96, 25.08, 26.11, 26.37,27.53, 28.18, 28.37, 30.33, 31.69, 33.75, 34.19, 36.23, 39.00, 39.36,39.81, 46.87, 47.64, 48.45, 54.91, 55.37, 55.48, 56.81, 57.08, 58.37,66.38, 69.14, 69.89, 71.42, 79.19, 82.66, 114.23, 130.36, 130.59,158.77, 168.43, 469.86, 170.72, 171.01, 171.21, 172.03, 173.62, 174.91.

ESI-MS Calcd for C₆₀H₉₉N₇O₁₄Si: 1169.7. Found m/z: 1170.9 (M+H)⁺.

Example 79 Synthesis of [Hiv]³-[D-Lac(OTBDMS)]⁹-aplidine [9LSHPL2(D)]

Following the procedure described for the synthesis of SAPL1, startingfrom SHPL2 (10 mg, 11.2 □mol) and (D)-Lac(OTBDMS)-Pro-OH (17 mg, 56□mol), the title compound (9 mg, 68%) was obtained as a white solidafter purification by HPLC (HyperPrep PEP 100 C18, gradient ACN/H₂O85:15-100:0 in 10 min (flow: 7 ml/min, 250×21 mm, at 270 nm, t_(R)=30.4min).

¹H NMR (300 MHz, CDCl₃) δ 0.03 (m, 3H), 0.06 (m, 3H) 0.87 (s, 9H),0.82-1.10 (m, 24H), 1.11-1.72 (m, 18H), 1.75-2.30 (m, 6H), 2.41 (m, 1H),2.56 (s, 3H), 3.00-3.40 (m, 5H), 3.06 (s, 3H), 3.56 (m, 1H), 3.65 (m,2H), 3.78 (s, 3H), 3.90 (m, 1H), 4.01 (m, 1H), 4.17 (m, 1H), 4.25 (m,1H), 4.39 (m, 1H), 4.61 (m, 2H), 4.86 (m, 1H), 5.00 (d, J=4.8, 1H), 5.25(m, 2H), 6.84 (d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.29 (d, J=9.7, 1H),7.74 (d, J=5.3, 1H), 7.87 (d, J=9.7, 1H).

¹³C NMR (75 MHz, CDCl₃) δ−4.87, −4.84, 12.04, 14.38, 16.89, 17.94,19.14, 20.24, 21.08, 21.60, 23.85, 24.12, 24.90, 25.06, 25.13, 26.01,26.53, 27.53, 27.83, 28.20, 30.33, 31.51, 33.76, 34.20, 36.16, 38.99,39.35, 39.82, 46.87, 47.02, 48.46, 54.79, 55.42, 55.48, 57.08, 57.18,58.28, 66.39, 69.11, 71.43, 72.61, 79.18, 114.25, 130.29, 130.59,158.79, 168.45, 169.86, 170.77, 170.81, 170.97, 171.20, 172.53, 173.67,174.84.

ESI-MS Calcd for C₆₀H₉₉N₇O₁₄Si: 1169.7. Found m/z: 1170.8 (M+H)⁺.

Example 80 Synthesis of [Val]³-[L-Lac(OTBDMS)]⁹-aplidine [9LSVPL2(L)]

Following the procedure described for the synthesis of SAPL1, startingfrom SVPL2 (10 mg, 11.2 □mol) and (L)-Lac(OTBDMS)-Pro-OH (17 mg, 56□mol), the title compound (9 mg, 68%) was obtained as a white solidafter purification by HPLC (HyperPrep PEP 100 C18, isocratic ACN/H₂O85:15 (flow: 7 ml/min, 250×21 mm, at 270 nm, t_(R)=17.8 min).

¹H NMR (300 MHz, CDCl₃): δ 0.14 (s, 6H), 0.71-1.06 (m, 27H), 1.10-1.42(m, 10H), 1.43-1.84 (m, 8H), 1.85-2.40 (m, 11H), 2.57 (s, 3H), 2.80 (d,J=14.7, 1H), 3.15 (s, 3H), 3.15-3.23 (m, 1H), 3.33-3.42 (m, 2H), 3.54(dd, J₁=4.2, J₂=10.8, 1H), 3.58-3.69 (m, 3H), 3.79 (s, 3H), 3.88-3.90(m, 1H), 4.05-4.12 (bt, 1H), 4.32 (bs, 1 h), 4.43-4.68 (m, 4H), 4.77 (t,J=10.5, 1H), 5.31-5.35 (m, 2H), 6.83 (d, J=8.4, 2H), 7.08 (d, J=8.7,2H), 7.39 (d, J=9.9, 1H), 7.45 (d, J=9.0, 1H), 7.60 (d, J=10.5, 1H),7.83 (d, J=4.5, 1H).

¹³C NMR (75 MHz, CDCl₃) δ−4.67, −4.49, 11.84, 14.13, 16.67, 18.19,18.41, 19.68, 20.48, 21.15, 21.40, 23.61, 23.92, 24.72, 24.80, 25.06,25.88, 26.24, 27.17, 28.04, 28.13, 29.78, 31.57, 33.33, 33.81, 35.99,38.84, 41.56, 41.95, 46.83, 47.52, 48.68, 54.09, 54.64, 55.26, 56.63,57.13, 58.36, 59.12, 66.20, 70.10, 70.57, 71.27, 77.20, 114.01, 130.13,130.36, 158.55, 168.09, 169.76, 170.07, 170.48, 170.77, 172.21, 172.33,173.58, 175.45.

ESI-MS Cald. for C₅₉94₀N₈O₁₄: 1168.72. Found (m/z): 1169.8 (M+H)⁺.

Example 81 Synthesis of [Hiv]³-[L-Lac]⁹-aplidine [9LSHPL1(L)]:Tamandarine A

To a solution of 9LSHPL2(L) (16 mg, 14 □mol) in THF (500 □ml, anh.) at0° C. under Ar, was added TBAF (50 □l, 1M in THF). After 1 h at 22° C.the mixture was concentrated in vacuo and the crude was purified byflash LC (silica gel, grad DCM:MeOH 1% to 5%) to yield the titlecompound 813 mg, 88%) as a white solid.

Experimental data were published: Fenical, W. et al., J. Org. Chem.2000, 65, 782-792.

¹H NMR (300 MHz, CDCl₃): δ0.82-0.96 (m, 18H), 1.02 (d, J=3.4, 3H), 1.04(d, J=3.4, 3H), 1.14-2.28 (m, 14H), 1.24 (s, 3H), 1.34 (d, J=6.8, 3H),1.43 (d, J=6.8, 3H), 2.44 (dd, J₁=7.8, J₂=17.1, 1H), 2.58 (s, 3H), 3.00(bs, 1H), 3.10 (s, 3H), 3.14-3.31 (m, 2H), 3.37-3.43 (m, 2H), 3.56-3.72(m, 5H), 3.79 (s, 3H), 3.90 (t, J=7.8, 1H), 4.02 (dt, J₁=3.4, J₂=9.8,1H), 4.25 (d, J=3.9, 1H), 4.30 (t, J=6.8, 1H), 4.37 (dd, J₁=7.3, J₂=8.3,1H), 4.65 (m, 1H), 4.71 (t, J=7.4, 1H), 4.87 (t, J=11.2, 1H), 5.03 (d,J=4.9, 1H), 5.29 (dd, J₁=3.4, J₂=11.7, 1H), 5.42 (m, 1H), 6.83 (d,J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.34 (d, J=9.8, 1H), 7.48 (d, J=5.4,1H), 7.76 (d, J=9.8, 1H).

ESI-MS Calcd for C₅₄H₈₅N₇O₁₄: 1055.6. Found: 1056.7 (M+H)⁺.

Example 82 Synthesis of [Hiv]³-[D-Lac]⁹-aplidine [9LSHPL1(D)]

Following the procedure described for the synthesis of 9LSHPL1(L),starting from 9LSHPL2(D) (20 mg, 17 □mol) and TBAF (50 □l, 1M in THF),afforded the little compound (14 mg, 78%) as a white solid, afterpurification by flash LC (silica gel, grad DCM:MeOH 1% to 5%).

¹H NMR (300 MHz, CDCl₃): δ 0.78-1.08 (m, 18H), 1.02 (d, J=3.9, 3H), 1.04(d, J=3.4, 3H), 1.10-2.36 (m, 14H), 1.20 (d, J=6.3, 3H), 1.34 (d, J=6.3,3H), 1.37 (d, J=6.3, 3H), 2.38-2.50 (dd, J₁=7.8, J₂=17.5, 1H), 2.56 (s,3H), 3.10 (s, 3H), 3.13-3.20 (m, 1H), 3.22-3.28 (m, 1H), 3.37-3.42 (dd,J₁=3.9, J₂=4.3, 1H), 4.61-4.68 (m, 3H), 3.69-3.76 (m, 3H), 3.77 (m, 1H),3.78 (s, 3H), 3.90 (t, J=7.8, 1H), 3.97-4.07 (m, 1H), 4.26 (m, 1H), 4.41(q, J=6.3, 1H), 4.63 (m, 1H), 4.71 (m, 1H), 4.86 (t, J=10.7, 1H), 5.01(d, J=4.8, 1H), 5.21-5.37 (m, 2H), 6.83 (d, J=8.3, 2H), 7.06 (d, J=8.3,2H), 7.41 (m, 2H), 7.77 (d, J=9.2, 1H).

ESI-MS Calcd for C₅₄H₈₅N₇O₁₄: 1055.62. Found: 1056.6 (M+H)⁺.

Example 83 Synthesis of [Val]³-[L-Lac]⁹-aplidine [9LSVPL1(L)]

Following the method described for the synthesis of 9LSHPL1, startingfrom 9LSVPL2 (5 mg, 4.3 □mol), afforded the little compound (4 mg, 88%)as a white solid, after purification by flash LC (silica gel, gradDCM:MeOH 1% to 5%).

¹H NMR (300 MHz, CDCl₃): δ 0.82-1.02 (m, 18H), 1.16-1.42 (m, 4H), 1.32(d, J=3.0, 3H), 1.40 (d, J=6.6, 3H), 1.56-1.83 (m, 8H), 1.95-2.34 (m,11H), 2.58 (s, 3H), 2.84 (d, J=14.7, 1H), 3.15 (s, 3H), 3.15-3.23 (m,1H), 3.36-3.42 (1H), 3.55 (dd, J₁=9.0, J₂=10.5, 2H), 3.64-3.66 (m, 3H),3.95 (dd, J₁=3.3, J₂=9.9, 1H), 4.08 (td, J₁=7.5, J₂=17.1, 1H), 4.32 (bs,1H), 4.41 (dd, J₁=6.6, J₂=9.9, 1H), 4.48 (dd, J₁=5.1, J₂=10.5, 1H), 4.61(dd, J₁=6.0, J₂=6.6, 1H), 4.69-4.80 (m, 2H), 5.29-5.35 (m, 1H), 5.57 (m,1H), 6.84 (d, J=8.1, 2H), 7.08 (d, J=8.7, 2H), 7.37 (d, J=3.9, 1H), 7.40(d, J=5.4, 1H), 7.60 (d, J=10.8, 1H), 7.72 (d, J=3.9, 1H).

ESI-MS Calcd for C₅₄H₈₆N₈O₁₃: 1054.63. Found: 1055.8 (M+H)⁺.

Synthesis of the spiro[4,4]nonane unit Example 84 Synthesis ofN-[(2R)-2-allyl-N-(tert-butoxycarbonyl)prolyl]D-leucine (9)

To a cooled (0° C.) solution of 8 (1.53 g, 6 mmol) in anh DCM (33 ml)under argon, was added: HOAt (980 mg, 7.2 mmol), D-Leu-OBn.pTsOH (2.65g, 12 mmol), NMM (1.21 g, 12 mmol) and DCC (1.48 g, 7.2 mmol). Themixture was stirred 2 h at 0° C. and then 12 h at r.t.; additionalD-Leu-OBn.pTsOH (0.66 g, 3 mmol) and NMM (0.30 g, 3 mmol) were added,and the mixture was stirred 3 h more. The mixture was filtered and thesolvent was concentrated in vacuo. The residue was dissolved in EtOAc(30 ml) and washed successively with NaHCO₃ (2×25 ml, sat), citric acid(2×25 ml, 10%) and brine (25 ml). The organic solution was dried(Na₂SO₄) and concentrated under reduced pressure. The residue waspurified by LC-silica (hex-EtOAc, 6:1) to afford 9 (2.63 g, 96%) as acolourless oil. [α]_(D) ²⁰ 12.4° (c 1, MeOH). HPLC [column □BondapackC₁₈ (Waters), 10 m, 3.9×300 mm, flow: 1 ml/min, at 214 nm, eluentCAN/0.05% TFA (40:60)] t_(R)=9.08 min].

¹H-NMR (300 MHz, DMSO-d₆) δ0.86 (m, 6H), 1.37 (s, 9H), 1.55-1.72 (m,5H), 2.00 (m, 2H), 2.64 (m, 1H), 2.86 (m, 1H), 3.15 (m, 1H), 3.55 (m,1H), 4.41 (m, 1H), 5.05-5.11 (m, 4H), 5.63-5.72 (m, 1H), 7.28-7.35 (m,5H).

¹³C-MNR (75 MHz, acetone-d₆) δ 21.6, 22.8, 24.6, 28.3, 34.7, 38.2, 41.3,49.38, 51.0, 66.9, 69.7, 80.1, 119.1, 128.3, 132.7, 153.9, 172.8.

Ref. Synthesis of 8: a) Seebach, D. et al. J. Am. Chem. Soc. 1983, 105,5390-5398. b) Genin, M. J. et al. J. Org. Chem. 1993, 58, 2334-2337.

Example 85 Synthesis of(5R,8RS)-1-(tert-butoxycarbonyl)-7-[(1R)-1-benzyloxycarbonyl-3-methylbutyl]-8-hydroxy-6-oxo-1,7-diazaspiro[4,4]nonane(10)

To a solution of 9 (1.56 g, 3.42 mmol) in MeOH/H₂O (2:1, 108 ml) underargon, a solution of OsO₄ (2.5% w/w, 2.9 ml) in tert-butanol was added,Stirring was continued for 10 nm in and NaIO₄ (2.195 g, 10.3 mmol) wasadded. After 24 h of stirring the reaction mixture diluted with H₂O (100ml) and extracted with EtOAc (3×50 ml). The combined organic extractswere washed with brine (50 ml), dried (Na₂SO₄) and concentrated underreduced pressure. The residue was purified by LC-silica (hex-EtOAc,gradient 80:20 to 0:100%) to afford diastereomers 10a and 10b (combined:1.17 g, 76%) as a white solid.

10a: HPLC [Column Novapack C18 (Waters), 3.9×150 mm, □□=1 ml/min, □=214nm, eluent: CH₃CN/0.05% TFA, (40/60)] t_(R)=14.45 min. m.p.: 140-141° C.[α]²⁰ _(D)−4° (c 1, MeOH).

¹H-NMR (300 MHz, acetone-d₆) δ 0.90 (m, 6H), 1.29 (s, 9H), 1.64-2.30 (m,9H); 2.68 (dd, 1H, J₁=6, J₂=13, 1H), 3.37 (m, 2H), 4.51 (dd, 1H), 5.13(d, J=15, 2H), 5.79 (t, J=5, 2H), 7.40 (m, 5H).

¹³C-MNR (75 MHz, acetone-d₆) δ 21.8, 23.8, 24.1, 24.9, 28.5, 39.6, 40.8,41.5, 48.5, 66.8, 79.4, 79.8, 81.2, 129.1, 129.3, 128.6, 171.7, 172.0.

ESI-MS: Calcd for C₂₅H₃₆N₂O₆: 460.26. Found m/z: 483.4 (M+Na)⁺.

10b: HPLC [Column Novapack C18 (Waters), 3.9×150 mm, □□=1 ml/min, □=214nm, eluent: CH₃CN/0.05% TFA, (40/60)] t_(R)=18.75 min. M.p.: 134-135° C.[α]²⁰ _(D)+26 (c 1.2, MeOH).

¹H-NMR (300 MHz, CDCl₃) δ 0.90 (6H, m), 1.40 (9H, s), 1.50-2.60 (9H, m),3.40 (2H, m), 4.20-5.40 (5H, m), 7.40 (5H, m).

¹³C-MNR (75 MHz, acetone-d₆) δ □21.3, 23.2, 24.1, 24.9, 28.3, 38.9,40.2, 42.5, 47.9, 53.2, 66.8, 77.5, 79.4, 80.6, 129.1, 171.2, 174.1.

ESI-MS Calcd for C₂₅H₃₆N₂O₆: 460.26. Found m/z: 483.5 (M+Na)⁺.

Example 86 Synthesis of(5R)-7-[(1R)-1-benzyloxycarbonyl-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonaneas trifluoracetate salt (11)

To a solution of 10 (430 mg, 0.93 mmol) in TFA (10 ml), NaBH₄ (106 mg,2.8 mmol) was added. The mixture was stirred for 2 h and then, thereaction was concentrated under reduced pressure. The residue waspartitioned between H₂O (5 ml) and DCM (20 ml). The organic phase wasdried (Na₂SO₄) and concentrated in vacuo to afford 11 as an orange oil(318 mg, quant.). [α]²⁰D+15 (c 1, MeOH).

¹H-NMR (300 MHz, acetone-d₆) 0.91 (m, 6H), 1.50 (m, 1H), 1.66-1.94 (m,2H), 2.11-2.72 (m, 6H), 3.48-3.72 (m, 4H), 4.74 (dd, J₁=6, J₂=15, 1H),5.18 (s, 2H), 7.37 (m, 5H).

¹³C-MNR (75 MHz, acetone-d₆) δ 21.2, 23.2, 23.9, 25.5, 30.2, 34.6, 38.0,42.2, 46.6, 53.9, 67.6, 69.6, 129.3, 161.1, 161.6, 170.9, 172.6.

ESI-MS Calcd for C₂₀H₂₈N₂O₃: 344.21. Found m/z: 345.3 (M+H)⁺.

Example 87 Synthesis of(5R)-1-(tert-butoxycarbonyl)-7-[(1R)-1-carboxy-3-methylbuty]-6-oxo-1,7-diazaspiro[4,4]nonane(12)

To a solution of 11 (150 mg, 0.44 mmol) in ACN (5 ml),tetramethylammonium hydroxyde pentahydrate (158 mg, 0.87 mmol) and Boc₂O(144 mg, 0.66 mmol) were added while stirring. After 6 h, additionalTMAH. 5 H₂O (158 mg) and Boc₂O (192 mg) were added. The reaction wasstirred for 2d and then, it was partitioned between H₂O (10 ml) and DCM(25 ml). The aqueous phase was liophilyzed and purified by LC-silica(DCM-MeOH, gradient 92:8 to 60:40) to yield 12 (100 mg, 64%) as a whitesolid.

Example 88 Synthesis of(5R)-1-(isobutyryl)-7-[(1R)-1-benzyloxycarbonyl-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane(13)

To a solution of 11 (169 mg, 0.49 mmol) in anh DCM (10 ml) at 0° C.under argon, were added TEA (199 mg, 1.96 mmol), DMAP (6 mg, 0.049 mmol)and dropwise isobutyryl chloride (104 mg, 0.98 mmol). The reactionmixture was allowed to warm to room temperature and stirred for 24 h.The crude was partitioned between H₂O (10 ml) and DCM (10 ml). Theorganic phase was washed with brine (10 ml), dried (Na₂SO₄) andconcentrated in vacuo. Pure compound 13 (150 mg, 74%) as a white solid,was obtained after LC-silica (hex-EtOAc, gradient 60:40 to 0:100).

HPLC [Column Novapack C₁₈ (Waters), 3.9×150 mm, □□=1 ml/min, □=214 nm,eluyente: CH₃CN/0.05% TFA, (50/50)] t_(R)=4.50 min. M.p.: 87° C. [α]²⁰_(D)+9.6 (c 1.4, MeOH).

¹H-NMR (300 MHz, CDCl₃) δ0.90 (2d, J=7, 6H), 1.09-1.12 (2d, 6H), 1.37(septuplet, J=6, 1H), 1.61-2.10 (m, 7H), 2.64 (m, 2H), 3.14 (dd, J₁=9,J₂=17, 1H), 3.64 (m, 3H), 4.85 (dd, J₁=5, J₂=10, 1H), 5.14 (d, J=6, 2H),7.32 (m, 5H).

¹³C-NMR (300 MHz, CDCl₃) δ 18.6, 18.7, 021.2, 23.1, 24.0, 24.8, 29.5,32.5, 35.7, 37.6, 40.5, 47.8, 52.7, 66.7, 76.3, 170.8, 174.3, 174.9.

ESI-MS Calcd for C₂₄H₃₄N₂O₄: 414.25. Found m/z: 415.4 (M+H)⁺.

Example 89 Synthesis of(5R)-1-(pyruvyl)-7-[(R)-1-benzyloxycarbonyl-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane(14)

Pyruvil chloride was prepared according to the method described in theliterature, Pansare, S. V.; Gnana R. R. “Asymmetric Allylation andreduction on an Ephedrine-Derived Template: Stereoselective Synthesis ofα-Hydroxy Acids and Derivatives” J. Org. Chem. 1998, 63, 4120-4124.α,α-dichloromethyl methyl ether (188 mg, 1.57 mmol) was added to pyruvicacid (115 mg, 1.31 mmol). The reaction mixture was stirred for 20 minand the resulting solution was warmed to 50-55° C. and then, stirred forfurther 30 min. The reaction mixture was allowed to cool to roomtemperature and DCM (3 ml) was added.

To a solution of 11 (150 mg, 0.33 mmol) in anh DCM (4 ml) at 0° C. underargon, were added TEA (200 mg, 1.98 mmol) and DMAP (4 mg, 0.033 mmol) tothe freshly solution of pyruvil chloride at 0° C. The reaction mixturewas allowed to warm to room temperature and stirred for 6 h. The crudewas washed successively with citric acid (5 ml, 10%), aq. NaHCO₃ sat. (5ml) and brine (5 ml). The organic phase was dried (Na₂SO₄) andconcentrated. Pure compound 14 (77 mg, 56%) as an oil, was obtainedafter LC-silica (hex-EtOAc, 1:3).

HPLC [Column Novapack C18 (Waters), 3.9×150 mm, □□=1 ml/min, □=214 nm,eluent: ACN/0.05% TFA, (50/50)] t_(R)=5.87 and 6.72 min.

¹H-MNR (200 MHz, CDCl₃) δ 0.92 and 0.95 (2d, J=6, 6H); 1.42 (m, 1H);1.61-2.39 (m, 7H), 2.44 (s, 3H), 2.77 (m, 1H), 3.22 (m, 1H), 3.56-3.78(m, 2H), 3.92 (m, 1H), 4.67 and 4.85 (dd, J₁=6, J₂=10, 1H), 5.21 (s,2H), 7.34 (m, 5H).

¹³C-MNR (75 MHz, CDCl₃) δ 21.2, 23.0, 24.4, 24.8, 26.4, 29.3, 35.6,37.3, 40.7, 48.9, 53.0, 66.8, 68.6, 135.0, 166.0, 170.8, 173.0, 198.0.

ESI-MS Calcd for C₂₃H₃₀N₂O₅: 414.22. Found m/z: 415.4 (M+H)⁺.

Example 90 Synthesis of(5R)-1-(2-methylacryloyl)-7-[(1R)-1-benzyloxycarbonyl-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane(15)

Following the procedure described for the synthesis of 13, starting from11 (200 mg, 0.43 mmol) and methylacryloyl chloride (89 mg, 0.86 mmol),the title compound (70 mg, 50%) was obtained as a colourless oil, afterpurification by LC (silica ge, hex-EtOAc, 2:1). HPLC [Column NovapackC₁₈ (Waters), 3.9×150 mm, □□=1 ml/min, □=214 nm, eluent: ACN/0.05% TFA,(25/75)] Rt=6.38 min.

¹H-MNR (200 MHz, CDCl₃) δ 0.91 (t, J=6, 6H), 1.44 (m, 1H), 1.64-1.93 (m,5H), 1.93 (s, 3H), 1.96-2.12 (m, 2H), 2.78 (m, 1H), 3.20 (m, 1H),3.56-3.68 (m, 3H), 4.80 and 4.77 (2d, J=10, 1H), 5.16 (s, 2H), 5.19 (d,J=9, 2H), 7.32 (m, 5H).

¹³C-MNR (75 MHz, CDCl₃) δ 19.8, 21.4, 23.0, 24.2, 24.9, 29.9, 37.1,37.7, 41.1, 50.2, 53.2, 66.6, 67.4, 116.9, 128.1, 128.3, 128.4, 135.0,141.7, 170.7, 174.1.

ESI-MS Calcd for C₂₄H₃₂N₂O₄: 412.24. Found: 413.3 (M+H)⁺.

Example 91 Synthesis of(5R)-1-(isobutiryl)-7-[(1R)-1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane(16)

A degassed solution of 13 (134 mg, 0.32 mmol) containing 10% Pd/C (27mg) was hydrogenated under 16 psi for 24 h. The mixture was filteredthrough a pad of celite and the filtered solution was concentrated underreduced pressure to afford 16 (100 mg, 95%) as a colourless oil. M.p.68-69° C. [α]²⁰ _(D)−2° (c 1.1, MeOH).

¹H-NMR [300 MHz, acetone-d₆] δ 0.87-0.91 (2d, J=7, 6H), 1.09-1.12 (2d,6H), 1.46 (m, 1H), 1.70 (m, 2H), 1.90-2.10 (m, 5H), 2.49 (m, 1H), 2.70(m, 1H), 3.30 (m, 1H), 3.49 (dt, J₁=8, J₂=10, 1H), 3.61-3.75 (m, 2H),4.71 (dd, J₁=5, J₂=11, 1H).

¹³C-NMR (75 MHz, CDCl₃) δ □16.4, 16.60□□21.1, 23.0, 24.3, 25.0, 30.8,32.6, 36.6, 36.9, 40.6, 48.0, 53.4, 67.5, 171.8, 174.2, 176.2.

ESI-MS Calcd for C₁₇H₂₈N₂O₄: 324.20. Found: 323.3 (M−1)⁺.

Example 92 Synthesis of(5R)-1-(pyruvil)-7-[(1R)-1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane(17)

A degassed solution of 14 (79 mg, 0.26 mmol) in methanol (20 ml)containing Pd—C (10%, 22 mg) was hydrogenated under atmospheric pressurefor 45 min. The filtered solution was concentrated under reducedpressure to afford 17 (79 mg, 95%) as a white solid. HPLC [ColumnNovapack C18 (Waters), 3.9×150 mm, □□=1 ml/min, □=214 nm, eluent:ACN/0.05% TFA, (20/80)] Rt=13.14 min.

¹H-MNR (200 MHz, CDCl₃) δ 0.93 (m, 6H), 1.43 (m, 1H), 1.71-2.22 (m, 7H),2.34 (s, 3H), 2.41 (s, 3H), 2.74 (m, 1H), 3.31 (m, 1H), 3.74 (m, 2H),3.92 (m, 1H), 4.80 (dd, J₁=6, J₂=10, 1H), 7.07 (bs, 1H).

¹³C-MNR (200 MHz, CDCl₃) δ 21.3, 23.2, 24.7, 25.1, 27.1, 29.9, 36.0,37.0, 41.0, 49.3, 53.6, 69.1, 162.1, 172.7, 173.7, 197.5.

ESI-MS Calcd for C₂₃H₃₀N₂O₅: 324.17. Found m/z: 325.1 (M+H)⁺.

Example 93 Synthesis of(5R)-1-(2-methylacryloyl)-7-[(1R)-1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane(18)

To a solution of 15 (65 mg, 0.16 mmol) in methanol (2.5 ml) were addedaq. NaOH (1.6 ml, 1N) and H₂O (1.6 ml) and the mixture was stirred atroom temperature for 1 h. The resulting mixture was concentrated atreduced pressure, and the residue was partitioned between H₂O (20 ml)and DCM (20 ml). The aqueous phase was acidified to pH=2 with aq HCl (10ml, 0.1 N) and extracted with DCM (3×20 ml). The combined organic phaseswere washed with brine (25 ml), dried (Na₂SO₄) and concentrated atreduced pressure to afford 18 (44 mg, 85%) as a white solid. HPLC[Column Novapack C18 (Waters), 3.9×150 mm, □□=1 ml/min, □=214 nm,eluent: ACN/0.05% TFA, (25/75)] Rt=6.38 min.

¹H-MNR (300 MHz, CDCl₃) δ 0.86 (d, J=5, 3H), 0.89 (d, J=5, 3H), 1.42 (m,1H), 1.55-2.25 (m, 8H), 2.56 (m, 1H), 3.19-3.44 (m, 2H), 3.57-3.67 (m,2H), 4.78 (d, J=11, 1H), 4.82 (d, J=11, 1H), 5.18 (d, J=6, 2H).

¹³C-MNR (75 MHz, CDCl₃) δ 19.5, 21.2, 23.1, 24.2, 25.1, 30.9, 36.5,37.6, 40.9, 50.5, 53.7, 67.5, 117.5, 140.6, 170.8, 174.2.

ESI-MS Calcd for C₁₇H₂₆N₂O₄: 322.19. Found: 323.2 (M+H)⁺.

Example 94 Synthesis of[(5R)-1-(tert-buthoxycarbonyl)-7-[(1R)-1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane]⁷⁻⁹-aplidine(9SBSAPL1)

Following the procedure described for the synthesis of SAPL3, startingfrom SAPL4 (10 mg, 13 □mol), 12 (5 mg, 14 □mol), HATU (12.4 mg), HOAt(4.5 mg), NMM (3.3 □l), DCM (140 □l) and DMF (70 □l), the title compound(11 mg, 73%) was obtained as a white solid after purification by HPLC(HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min, 250×21mm, at 270 nm, Rt=30 min).

¹H NMR (300 MHz, CDCl₃): δ 0.85-0.97 (m, 24H), 1.19-1.34 (m, 18H), 1.48(s, 9H), 1.50-2.20 (m, 6H), 2.32-2.36 (m, 1H), 2.54 (s, 3H), 2.58-2.72(m, 2H), 2.97-3.08 (m, 1H), 3.10-3.22 (m, 3H), 3.34 (dd, 1H, J=3.9,13.8), 3.46-3.79 (m, 6H), 3.79 (s, 3H), 4.03-4.12 (m, 2H), 4.28 (dd, 1H,J=6.6, 13.2), 4.57-4.63 (m, 2H), 4.79-4.88 (m, 2H), 5.15 (d, 1H, J=3.3),5.21-5.23 (m, 1H), 6.84 (d, 2H, J=8.4), 7.07 (d, 2H, J=8.7), 7.28 (d,1H, J=10.8), 7.79 (d, 1H, J=6.6), 7.82 (d, 1H, J=9.9).

¹³C NMR (75 MHz, CDCl₃) δ 11.66, 15.21, 15, 43, 16.79, 17.23, 18.69,18.77, 21.24, 23.67, 23.95, 24.10, 24.90, 25.10, 25.38, 27.04, 28.23,28.75, 29.93, 31.61, 34.50, 33.88, 34.16, 36.20, 36.57, 38.76, 39.09,39.78, 41.29, 47.27, 47.76, 49.60, 49.96, 52.66, 55.50, 56.26, 57.38,58.18, 66.74, 66.86, 68.34, 70.53, 80.75, 81.93, 114.33, 130.23, 130.54,168.08, 169.83, 170.18, 170.80, 171.43, 172.55, 175.04, 205.04.

ESI-MS Calcd for C₆₀H₉₃N₇O₁₅ 1151.7. Found m/z: 1152.4 (M+H)⁺

Example 95 Synthesis of[Hiv]³-[(5R)-1-(tert-buthoxycarbonyl)-7-[1R)-1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane]⁷⁻⁹-aplidine

Following the procedure described for the synthesis of SAPL3, startingfrom SHPL4 (10 mg, 13 □mol), 12 (5 mg, 14 □mol), HATU (14 mg), HOAt (5mg), NMM (6 □l), DCM (150 □l) and DMF (50 □l), the title compound (10mg, 70%) was obtained as a white solid after purification by HPLC(HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min, 250×21mm, at 270 nm, Rt=28.1 min).

¹H NMR (300 MHz, CDCl₃) δ 0.80-1.07 (m, 24H), 1.08-1.67 (m, 12H), 1.48(s, 9H), 1.68-2.30 (m, 10H), 2.41 (m, 1H), 2.55 (s, 3H), 2.68 (m, 1H),2.94 (m, 1H), 3.07-3.40 (m, 4H), 3.42-3.72 (m, 6H), 3.78 (s, 3H), 3.90(m, 1H), 4.01 (m, 1H), 4.29 (m, 1H) 4.63 (m, 1H), 4.77 (m, 1H), 4.87 (m,1H), 5.02 (d, J=4.8, 1H), 5.25 (m, 1H), 6.84 (d, J=8.3, 2H), 7.07 (d,J=8.3, 2H), 7.31 (d, J=9.7, 1H), 7.50 (d, J=5.8, 1H), 7.85 (d, J=9.7,1H).

¹³C NMR (75 MHz, CDCl₃) δ 12.07, 14.25, 17.12, 17.92, 19.15, 21.11,21.18, 23.80, 24.00, 24.06, 24.86, 25.08, 25.15, 27.58, 28.21, 28.82,30.33, 31.18, 33.78, 34.28, 35.77, 36.65, 39.07, 39.45, 39.86, 46.91,48.14, 48.47, 52.62, 55.49, 57.13, 58.42, 66.36, 66.80, 69.14, 70.84,79.19, 80.55, 114.27, 130.29, 130.59, 154.12, 158.81, 168.25, 169.84,170.72, 170.80, 170.90, 171.25, 174.89.

ESI-MS Calcd for C₅₇H₈₉N₇O₁₄ 1095.6. Found m/z: 1096.9 (M+H)⁺.

Example 96 Synthesis of[(5R)-1-(isobutiryl)-7-[(R)-1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane]⁷⁻⁹-Aplidine(9SISAPL1)

Following the procedure described for the synthesis of SAPL3, startingfrom SAPL4 (11 mg, 12.9 □mol), 16 (5 mg, 15.4 □mol), HATU (14 mg), HOAt(5 mg), NMM (3.6 □l), DCM (155 □l) and DMF (78 □l), the title compound(10 mg, 69%) was obtained as a white solid after purification by HPLC(HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min, 250×21mm, at 270 nm, t_(R)=19 min).

¹H NMR (300 MHz, CDCl₃): δ0.86-1.00 (m, 24H), 1.12 (d, J=6.9, 3H), 1.18(d, J=6.6, 3H), 1.34 (t, J=6.6, 2H), 0.90-1.30 (m, 7H), 1.56-2.25 (m,16H), 2.30-2.80 (m, 3H), 2.55 (s, 3H), 2.95-3.06 (m, 1H), 3.15-3.25 (m,3H), 3.65 (dd, 1H) 3.52-3.79 (m, 6H), 3.79 (s, 3H), 3.98-4.15 (m, 1H),4.28 (dd, J₁=6.6, J₂=10.3, 1H), 4.59 (m, 2H), 4.79-4.85 (m, 2H), 5.17(d, J=3.6, 1H), 5.40-5.44 (m, 1H), 6.84 (d, J=8.4, 2H), 7.06 (d, J=8.7,2H), 7.24 (d, J=11.1, 1H), 7.90 (d, J=9.3, 1H), 8.56 (d, J=5.1, 1H).

¹³C NMR (75 MHz, CDCl₃) δ: 11.50, 14.92, 15.22, 16.68, 16.95, 18.50,18.59, 18.81, 20.94, 23.42, 23.80, 24.49, 24.68, 24.90, 25.11, 26.91,27.98, 30.93, 31.30, 35.58, 33.88, 34.16, 35.85, 36.24, 38.64, 38.84,39.71, 41.29, 47.01, 47.76, 49.42, 49.62, 52.66, 55.26, 55.73, 57.12,58.21, 66.57, 67.40, 68.10, 70.79, 81.57, 114.07, 130.05, 130.31,158.57, 168.12, 169.66, 170.08, 170.56, 171.13, 171.96, 172.37, 174.04,175.41, 205.04.

ESI-MS Calcd for C₅₉H₉₁N₇O₁₄: 1121.66. Found: 1122.8 (M+H)⁺.

Example 97 Synthesis of[Hiv]³-[(5R)-1-(tert-buthoxycarbonyl)-7-[(1R)-1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane]⁷⁻⁹-aplidine(9SISHPL1)

Following the procedure described for the synthesis of SAPL5, startingfrom SHPL4 (10 mg, 13 □mol), 16 (4.5 mg, 14 □mol), HATU (14 mg), HOAt (5mg), NMM (6 □l), DCM (150 □l) and DMF (50 51), the title compound (10mg, 72%) was obtained as a white solid after purification by HPLC(HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min, 250×21mm, at 270 nm, t_(R)=16.9 min).

¹H NMR (300 MHz, CDCl₃) δ 0.80-1.07 (m, 24H), 1.08-1.47 (m, 12H),1.48-2.30 (m, 16H), 2.36 (m, 2H), 2.56 (s, 3H), 2.65 (m, 1H), 2.96 (m,1H), 3.18 (m, 2H), 3.36 (m, 2H), 3.65 (m, 6H), 3.78 (s, 3H), 3.91 (m,1H), 4.02 (m, 1H), 4.25 (m, 1H) 4.63 (m, 1H), 4.72 (m, 2H), 4.87 (m,1H), 5.02 (d, J=4.8, 1H), 5.45 (m, 1H), 6.84 (d, J=8.7, 2H), 7.07 (d,J=8.7, 2H), 7.27 (d, J=4.8, 1H), 7.88 (d, J=9.7, 1H), 8.32 (d, J=4.8,1H).

ESI-MS Calcd for C₅₆H₈₇N₇O₁₃ 1065.6. Found m/z: 1066.7 (M+H)⁺.

Example 98 Synthesis of[(5R)-1-(pyruvyl)-7-[(1R)-1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane]⁷⁻⁹-aplidine(9SPSAPL1)

Following the procedure described for the synthesis of SAPL3, startingfrom SAPL4 (10 mg, 11.7 □mol), 17 (5 mg, 15.4 □mol), HATU (12 mg), HOAt(5 mg), NMM (5 □l), DCM (140 □l) and DMF (70 □l), the title compound (9mg, 63%) was obtained as a white solid after purification by HPLC(HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min, 250×21mm, at 270 nm, t_(R)=14.4 min).

¹H NMR (300 MHz, CDCl₃): δ 0.90-1.00 (m, 24H), 1.05-1.40 (m, 12H),1.40-2.25 (m, 16H), 2.27-2.41 (m, 1H), 2.42-2.70 (m, 3H), 2.54 (s, 3H),2.92-2.98 (m, 1H), 3.12-3.38 (m, 4H), 3.54-3.78 (m, 4H), 3.79 (s, 3H),4.01-4.12 (m, 2H), 4.20-4.26 (m, 2H), 4.57-4.62 (m, 2H), 4.77-4.82 (m,2H), 535.18 (d, J=3.0, 2H), 5.37-5.42 (m, 1H), 6.84 (d, J=8.7, 2H), 7.07(d, J=8.7, 2H), 7.20 (d, J=9.6, 1H), 7.85 (d, J=9.6, 1H), 98.04 (d,J=54, 14H).

¹³C NMR (75 MHz, CDCl₃) δ 11.86, 14.97, 15.47, 16.78, 17.12, 18.84,21.20, 21.27, 23.62, 24.14, 15.03, 25.15, 25.30, 27.35, 27.51, 28.19,30.46, 31.52, 34.27, 35.95, 39.01, 40.07, 41.59, 47.25, 49.72, 53.08,55.51, 55.81, 57.38, 58.21, 66.66, 68.19, 69.23, 70.74, 81.69, 85.15,114.33, 130.13, 130.56, 158.85, 161.12, 168.49, 169.81, 169.91, 170.81,171.38, 171.69, 172.60, 173.34, 197.64, 205.19.

ESI-MS Calcd for C₅₈H₈₇N₇O₁₅: 1121.63. Found 1122.3 (M+H)⁺.

Example 99 Synthesis of[Hiv]³-[(5R)-1-(pyruvyl)-7-[(1R)-1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane]⁷⁻⁹-aplidine(9SPSHPL1)

Following the procedure described for the synthesis of SAPL3, startingfrom SHPL4 (10 mg, 13 □mol), 17 (4.5 mg, 14 □mol), HATU (14 mg), HOAt (5mg), NMM (6 □l), DCM (150 □l) and DMF (50 □l), the title compound (10mg, 72%) was obtained as a white solid after purification by HPLC(HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min, 250×21mm, at 270 nm, t_(R)=13.6 min).

¹H NMR (300 MHz, CDCl₃) δ 0.80-1.10 (24H, m), 1.11-1.80 (12H, m),1.81-2.30 (10H, m), 2.45 (m, 1H), 2.55 (s, 3H), 2.57 (s, 3H), 3.07-3.43(m, 6H), 3.52-3.77 (m, 6H), 3.78 (s, 3H), 3.91 (m, 1H), 4.03 (m, 1H),4.29 (m, 1H) 4.63 (m, 1H), 4.72 (m, 1H), 4.87 (m, 1H), 5.03 (d, J=4.3,1H), 5.45 (m, 1H), 6.84 (d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.29 (d,J=8.7, 1H), 7.81 (d, J₁=9.2, 1H), 7.87 (d, J=4.8, 1H).

ESI-MS Calcd for C₅₅H₈₃N₇O₁₄: 1065.6. Found 1066.4 (M+H)⁺.

Example 100 Synthesis of[Hiv]³-[(5R)-1-(acriloyl)-7-[(1R)-1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4,4]nonane]⁷⁻⁹-aplidine(9SASHPL1)

Following the procedure described for the synthesis of SAPL3, startingfrom SHPL4 (10 mg, 13 □mol), 18 (5.8 mg, 14 □mol), HATU (14 mg), HOAt (5mg), NMM (6 □l), DCM (150 □l) and DMF (50 □l), the title compound (10mg, 72%) was obtained as a white solid after purification by HPLC(HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min, 250×21mm, at 270 nm, t_(R)=16.4 min).

¹H NMR (300 MHz, CDCl₃) δ 0.85-0.96 (m, 18H), 1.02-1.05 (m, 6H),1.14-1.45 (m, 12H), 1.49-1.64 (m, 4H), 1.68-1.77 (m, 1H), 1.89-2.05 (m,3H), 1.99 (s, 3H), 2.10-2.28 (m, 4H), 2.43 (dd, J₁=7.8, J₂=17.1, 1H),2.57 (s, 3H), 2.60-2.68 (m, 1H), 2.97 (bs, 1H), 3.13-3.40 (m, 4H),3.54-3.77 (m, 5H), 3.79 (s, 3H), 3.89-4.07 (m, 2H), 4.27 (m, 1H), 4.64(m, 1H), 4.73 (m, 1H), 4.88 (m, 1H), 5.03 (d, J=4.4, 1H), 5.30 (d, J=20,1H), 5.30-5.39 (m, 1H), 6.84 (d, J=8.3, 2H), 7.08 (d, J=8.3, 2H), 7.29(s, 1H), 7.88 (d, J=9.8, 1H), 8.23 (d, J=7.4, 1H).

ESI-MS Calcd for C₅₆H₈₅N₇O₁₃: 1063.6. Found 1064.6 (M+H)⁺.

Example 101 Synthesis of [Hiv]³-[Z-Ala]⁹-aplidine (9ZASHPL2)

To a flask containing Z-Ala-Pro-OH (36 mg, 112 □mol) in DCM (0.4 ml) at0° C., under argon, DIPCDI (10 □l, 64 □mol) was added and the mixturewas stirred for 60 min. Then, a solution of SHPL2 (20 mg, 22.5 □mol) inDCM (0.2 ml) was added and after 3 d the reaction was quenched byaddition of aq HCl (3 ml, 0.1 N). The mixture was stirred for 5 min andthen, diluted with DCM (4 ml) and washed successively with aq. KHSO₄ (4ml, 10%), aq. NaHCO₃ (4 ml, sat) and brine (4 ml). The organic phase wasdried (Na₂SO₄), filtered and concentrated. Purification of the residueby HPLC (HyperPrep PEP 100 C18, isocratic ACN/H₂O 85:15 (flow: 7 ml/min,250×21 mm, at 270 nm, t_(R)=18.2 min) afforded 9ZASHPL2 (30 mg, 67%) asa white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.10 (m, 24H), 1.12-1.50 (m, 18H),1.52-2.70 (m, 6H), 2.45 (m, 1H), 2.56 (s, 3H), 2.96-3.38 (m, 4H), 3.10(s, 3H), 3.52-3.72 (m, 5H), 3.85 (m, 1H), 3.78 (s, 3H), 4.01 (m, 1H),4.18 (m, 1H), 4.51 (m, 1H), 4.64 (m, 1H), 4.71 (m, 1H), 4.87 (m, 1H),5.02 (d, J₁=5.3, 1H), 5.06 (m, 2H), 5.25 (m, 1H), 5.42 (m, 1H), 6.10 (d,J=8.3, 1H), 6.84 (d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.31 (m, 6H), 7.70(d, J=4.3, 1H), 7.77 (d, J=9.7, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.80, 13.97, 16.63, 17.02, 17.48, 18.93,20.86, 21.15, 23.45, 23.76, 24.78, 25.96, 27.31, 27.92, 28.50, 30.06,31.36, 33.44, 33.82, 35.66, 38.70, 39.38, 39.63, 46.54, 46.62, 46.98,47.10, 48.19, 48.59, 54.89, 54.97, 55.22, 56.44, 56.90, 58.14, 66.16,66.36, 68.95, 70.94, 78.78, 114.00, 127.75, 128.30, 129.90, 130.26,156.31, 158.53, 168.67, 169.57, 170.06, 170.25, 170.65, 171.97, 173.08,174.67.

ESI-MS Calcd for C₆₂H₉₂N₈O₁₅ 1188.67. Found m/z 1189.7 (M+H)⁺.

Example 102 Synthesis of [Hiv]³-[Boc-Ala]⁹-aplidine (9BASHPL2)

Following the procedure described for the synthesis of 9ZASHPL2, fromSHPL2 (10 mg, 11.2 □mol), Boc-Ala-Pro-OH (17 mg, 57 □mol), DIPCDI (5 □l)and DCM (300 □l), the title compound (9 mg, 70%) was obtained as a whitesolid after purification by HPLC (HyperPrep PEP 100 C18, gradientACN/H₂O 85:15-100:0 in 10 min (flow: 7 ml/min, 250×21 mm, at 270 nm,t_(R)=14.5 min).

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.10 (m, 24H), 1.12-1.50 (m, 15H), 1.40(s, 9H), 1.52-2.70 (m, 9H), 2.45 (m, 1H), 2.60 (s, 3H), 3.00-3.43 (m,4H), 3.10 (s, 3H), 3.62 (m, 5H), 3.79 (s, 3H), 3.90 (m, 1H), 4.02 (m,1H), 4.20 (m, 1H), 4.41 (m, 1H), 4.67 (m, 2H), 4.87 (m, 1H), 5.02 (d,J₁=4.3, 1H), 5.26 (m, 1H), 5.40 (m, 1H), 5.76 (d, J=7.8, 1H), 6.84 (d,J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.32 (d, J=9.7, 1H), 7.73 (d, J=4.8,1H), 7.80 (d, J=9.7, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 12.06, 14.34, 16.88, 17.20, 17.77, 19.17,21.10, 21.41, 23.72, 24.03, 25.06, 26.17, 27.60, 28.19, 28.60, 30.31,31.63, 33.74, 35.96, 38.96, 38.93, 39.59, 39.86, 46.86, 47.27, 48.29,48.45, 55.18, 55.42, 55.49, 56.59, 57.16, 58.44, 66.43, 69.14, 71.30,79.05, 79.40, 114.29, 130.21, 130.54, 156.06, 158.83, 168.80, 169.87,170.51, 170.64, 170.94, 171.31, 172.59, 173.45, 174.92.

ESI-MS Calcd for C₅₉H₉₄N₈O₁₅ 1154.68. Found m/z 1155.6 (M+H)⁺.

Example 103 Synthesis of [Hiv]³-[Ala]⁹ aplidine (9ASHPL1)

To a flask containing 9BASHPL2 (8 mg, 6.92 □mol), a solution ofhydrochloric acid in anh. dioxane (1.5 ml, 5.3 N, 7.9 mmol) was added.The resulting solution was stirred at room temperature for 5 h or untilcomplete disappearance of the starting material (TLC). Then, thesolution was concentrated under reduced pressure and the residue wasdissolved in DCM and concentrated again. The white foam crude wasprecipitated with DCM/hex (2 ml/4 ml) to yield 9ASHPL1 (7.2 mg, quant.)as a white solid.

ESI-MS Calcd for C₅₄H₈₆N₈O₁₃ 1054.6. Found m/z: 1055.6 (M+H)⁺.

Example 104 Synthesis of [Hiv]³-[Boc-Pro]⁸-didemnin A (8PSHPL2)

Following the procedure described for the synthesis of 9ZASHPL2,starting from SHPL2 (10 mg, 11.2 □mol), Boc-Pro-OH (13 mg, 57 □mol),DIPCDI (5 □l) and DCM (300 □l), the title compound (9 mg, 74%) wasobtained as a white solid after purification by HPLC (HyperPrep PEP 100C18, gradient ACN/H₂O 85:15-100:0 in 10 min (flow: 7 ml/min, 250×21 mm,at 270 nm, t_(R)=18.7 min).

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.10 (m, 24H), 1.12-2.30 (m, 22H), 1.47(s, 9H), 2.41 (m, 1H), 3.04 (s, 3H), 3.10-3.74 (m, 7H), 3.78 (s, 3H),3.91 (m, 1H), 4.01 (m, 1H), 4.31 (m, 1H), 4.59 (m, 1H), 4.87 (m, 1H),5.02 (d, J=4.8, 1H), 5.16 (m, 1H), 5.36 (m, 1H), 6.84 (d, J=8.3, 2H),7.07 (d, J=8.3, 2H), 7.17 (d, J=6.3, 1H), 7.35 (d, J=9.7, 1H), 7.85 (d,J=9.7, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 12.04, 14.41, 16.76, 17.97, 19.12, 21.03,21.66, 23.92, 24.00, 24.77, 25.09, 25.20, 27.53, 28.21, 28.72, 29.67,30.35, 31.23, 33.80, 34.30, 36.24, 39.05, 39.37, 39.80, 46.90, 47.33,48.42, 54.48, 55.49, 55.58, 55.84, 57.12, 58.08, 66.31, 69.11, 71.05,79.23, 80.28, 114.29, 130.23, 130.59, 154.91, 158.82, 168.37, 169.88,170.66, 170.86, 171.28, 171.40, 174.10, 174.79.

ESI-MS Calcd for C₅₆H₉₁N₇O₁₄; 1085.6. Found m/z; 1086.7 (M+H)⁺.

Example 105 Synthesis of [Hiv]³-[Pro]⁸-didemnin A (8PSHPL1)

Following the procedure described for the synthesis of 9ASHPL1, startingfrom 8PSHPL2 (7 mg, 6.4 □mol), the title compound (6 mg, quant.) wasobtained as a white solid.

ESI-MS Calcd for C₅₁H₈₁N₇O₁₂: 983.59. Found m/z: 984.6 (M+H)⁺.

Example 106 Synthesis of [Hiv]³-[Boc-Val]⁸-didemnin A (8VSHPL2)

Following the procedure described for the synthesis of 9ZASHPL2,starting from SHPL2 (10 mg, 11.2 □mol), Boc-Val-OH (12 mg, 56 □mol),DIPCDI (5 □l) and DCM (300 0 □l), the title compound (9 mg, 82%) wasobtained as a white solid after purification by HPLC (HyperPrep PEP 100C18, gradient ACN/H₂O 85:15-100:0 in 10 min (flow: 7 ml/min, 250×21 mm,at 270 nm, t_(R)=22.1 min).

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.10 (m, 30H), 1.12-1.82 (m, 12H), 1.45(s, 9H), 1.84-2.40 (m, 6H), 2.56 (s, 3H), 2.96 (s, 3H), 2.97 (m, 3H),3.13 (m, 1H), 3.35 (m, 1H), 3.56 (m, 1H), 3.65 (m, 2H), 3.79 (s, 3H),3.85 (m, 1H), 4.03 (m, 1H), 4.24 (m, 1H), 4.41 (m, 1H), 4.61 (m, 1H),4.88 (m, 1H), 4.99 (d, J₁=5.3, 1H), 5.07 (m, 1H), 5.19 (m, 1H), 5.60 (d,J=8.3, 1H), 6.84 (d, J=8.3, 2H), 7.03 (d, J=8.7, 2H), 7.07 (d, J=8.3,2H), 7.46 (d, J=10.2, 1H), 7.84 (d, J=9.2, 1H).

ESI-MS Calcd for C₅₆H₉₁N₇O₁₄: 1085.6. Found m/z: 1086.7 (M+H)⁺.

Example 107 Synthesis of [Hiv]³-[Val]⁸-didemnin A (8VSHPL1)

Following the procedure described for the synthesis of 9ASHPL1, startingfrom 8VSHPL2 (8 mg, 7.4 □mol), the title compound (7 mg, quant.) wasobtained as a white solid.

ESI-MS Calcd for C₅₁H₈₃N₇O₁₂: 985.61. Found: (m/z): 986.6 (M+H)⁺.

Example 108 Synthesis of [Hiv]³-[Val]⁸-[Isobutyryl]⁹-didemnin A(8V9ISHPL1)

To a solution of 8VSHPL1 (6 mg, 5.8 □mol) in DCM (200 0 ml) at 0° C.under Ar, were added NMM (3.3 □l, 30 □mol) and isobutyryl chloride (2□l, 19 □mol). After 5 h of stirring at r.t., the reaction mixture wasdiluted with DCM (5 ml) and washed successively with aq. KHSO₄ (5 ml,10%), aq. HCO₃Na (5 ml, sat) and brine (5 ml). The organic solution wasdried (Na₂SO₄), filtered and concentrated at reduced pressure to yield8V91SHPL1 (6 mg, 97%) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.10 (m, 36H), 1.12-1.85 (m, 10H),1.90-2.45 (m, 6H), 2.56 (s, 3H) 2.90 (m, 1H), 3.00 (s, 3H), 3.13 (m,2H), 3.36 (dd, J₁=4.3, J₂=14.1, 1H), 3.64 (m, 6H), 3.78 (s, 3H), 3.87(m, 1H), 3.97 (m, 1H), 4.21 (m, 1H), 4.34 (m, 2H), 4.61 (m, 1H), 4.87(m, 1H), 4.98 (d, J=4.8, 1H), 5.17 (m, 2H), 6.24 (d, J=6.3, 1H), 6.84(d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.32 (d, J=6.3, 1H), 7.35 (d,J=4.3, 1H), 7.81 (d, J=9.7, 1H).

ESI-MS Calcd for C₅₅H₈₉N₇O₁₃, 1055.6. Found m/z: 1056.7 (M+H)⁺.

Example 109 Synthesis of [coumarin]⁸-didemnin A (8CSAPL1)

Following the procedure described for the synthesis of 9ZASHPL2,starting from SAPL2 (20 mg, 21 □mol) and coumarin-3-carboxylic acid (20mg, 107 □mol), the title compound (18 mg, 76%) was obtained as a whitesolid after purification by HPLC (HyperPrep PEP 100 C18, isocraticACN/H₂O 85:15 (flow: 7 ml/min, 250×21 mm, at 270 nm, t_(R)=16.5 min).

¹H NMR (300 MHz, CDCl₃) δ 0.78-1.00 (m, 24H), 1.20-2.50 (m, 22H), 2.56(s, 3H), 2.92 (s, 3H), 3.08-3.25 (m, 2H), 3.90 (m, 1H), 3.60 (m, 2H),3.70 (m, 1H), 3.79 (s, 3H), 3.92-4.25 (m, 3H), 4.60 (m, 1H), 4.80 (m,2H), 5.15 (m, 1H), 5.18 (d, J=3.4, 1H), 5.28 (m, 1H), 6.84 (d, J=8.3,2H), 7.08 (d, J=8.3, 2H), 7.20 (d, J=6.3, 1H), 7.37 (m, 2H), 7.45 (d,J=9.7, 1H), 7.58 (m, 2H), 7.96 (m, 1H), 8.23 (m, 1H).

ESI-MS Calcd for C₅₉H₈₂N₆O₁₅: 1114.58. Found: 1116.3(M+H)⁺.

Example 110 Synthesis of coumarin-3-carbonylamino-acetic acid methylester (8G9C2)

To a round bottom flask containing methyl-glycine (89 mg, 1.00 mmol),3-carboxy coumarine, anh. DCM (25 ml), under Ar, N′-(3Dimethylaminopropil)-N-ethyl-carbodiimid hydrochlorid (EDC) (479 mg,2.50 mmol) and DMAP (489 mg, 4.00 mmol) were added at room temperature.The resulting mixture was stirred for 1 h 30 min (total conversion wasobserved by TLC). Then, DCM (20 ml) was added and the solution waswashed successively with aq. NaHCO₃ (10 ml, sat) and brine (10 ml). Theorganic phase was dried (Na₂SO₄), filtered and concentrated. Theresulting orange solid obtained was purified by flash LC (silica gel,grad hex:EtOAc 1:1 to 2:1) to give the title compound (445 mg, quant) asa colourless oil. Rf=0.08 (Hex/EtOAc 1:1).

¹H NMR (300 MHz, CDCl₃) δ 3.79 (s, 3H), 4.25 (d, J=5.4, 2H), 7.40 (m,2H), 7.74 (d, J=7.3, 1H), 7.68 (m, 2H), 8.91 (s, 1H), 9.25 (s, 1H).

ESI-MS Calcd for C₁₃H₁₁NO₅: 261.06. Found: 283.1 (M+Na)⁺.

Example 111 Synthesis of coumarin-3-carbonylamino-acetic acid (8G9C1)

A solution of coumarin-3-carbonylamino-acetic acid methyl ester (312 mg,1.19 mmol), in THF (12 ml) under Ar atmosphere, at 0° C. (ice bath), asolution of LiOH in H₂O (0.2 M) was added dropwise. The reaction mixturewas stirred vigorously at room temperature until total convertion wasobserved by TLC (2 hours). The solution was partially concentrated andEt₂O was added (10 ml). The organic layer was washed with NaHCO₃ (10 ml,sat) and the combined aqueous layers were acidified with 10% KHSO₄(pH=3-4) and extracted with ether (3×20 ml). The organic layer wasconcentrated at reduced pressure to afford 8G9C1 (280 mg, 95%) as awhite solid.

¹H NMR (300 MHz, CDCl₃) 3 4.18 (d, J=5.3, 2H), 7.41 (m, 1H), 7.45 (d,J=7.8, 1H), 7.74 (d, J=7.3, 1H), 7.78 (t, J=8.3, 1H), 7.85 (d, J=7.8,1H), 8.89 (s, 1H), 9.41 (m, 1H).

ESI-MS Calcd for C₁₂H₉NO₅: 247.05. Found: 248.0 (M+H)⁺.

Example 112 Synthesis of [Gly]⁸-[coumarin]⁹-didemnin A (8G9CSAPL1)

Following the procedure described for the synthesis of 9ZASHPL2,starting from SAPL2 (20 mg, 21 □mol) and coumarin-3-carbonylamino-aceticacid (26 mg, 105 □mol), the title compound (18 mg, 72%) was obtained asa white solid after HPLC (Symmetry Prep™ C18, isocratic ACN/H₂O 60:40(flow: 3 ml/min, 150×7.8 mm, at 270 nm, t_(R)=17.5 min).

¹H NMR (300 MHz, CDCl₃) δ 0.85-0.94 (m, 24H), 1.23-2.17 (m, 21H),2.30-2.44 (m, 1H), 2.56 (s, 3H), 3.01 (s, 3H), 3.10-3.24 (m, 2H), 3.40(dd, J₁=5.4, J₂=14.2, 1H), 3.59-3.74 (m, 3H), 3.79 (s, 3H), 3.99-4.21(m, 4H), 4.37-4.44 (m, 1H), 4.60 (m, 1H), 4.68 (m, 1H), 4.81 (t, J=9.8,1H), 5.18 (d, J=3.4, 1H), 5.25 (dd, J₁=2.9, J₂=5.9, 1H), 5.20-5.45 (m,1H), 6.84 (d, J=8.3, 2H), 7.08 (d, J=8.3, 2H), 7.19-7.28 (m, 1H),7.34-7.42 (m, 2H), 7.63-7.72 (m, 2H), 7.88 (d, J=8.3, 2H), 9.00 (s, 1H),9.57 (m, 1H).

ESI-MS Calcd for C₆₁H₈₅N₇O₁₆: 1171.61. Found: 1172.5 (M+H)⁺.

Example 113 Synthesis of N-methylsulphonyl-Pro-OBz (P2)

To a solution of Pro-OBn (HCl) (300 mg, 1.24 mmol) in DCM (25 ml, anh)at 0° C. under Ar, DIPEA (0.7 □l) and methanesulphonyl chloride (116 □l)were added dropwise by syringe. The reaction mixture was stirred at roomtemperature overnight. DCM (10 ml) was added and the solution was washedsuccessively with aq. KHSO₄ (15 ml, 10%), aq. NaHCO₃ (15 ml, sat) andbrine (15 ml). The organic phase was dried (Na₂SO₄), filtered andconcentrated to reduce pressure to yield pure P2 (350 mg, 1.24 mmol,quant) as a white solid. Rf=0.55 (Hex/AcOEt 1; 1).

¹H NMR (300 MHz, CDCl₃) δ 1.91-2.08 (m, 3H), 2.19-2.31 (m, 1H), 2.93 (s,3H), 3.38-3.54 (m, 2H), 3.65 (s, 3H), 4.51 (dd, J₁=3.4, J₂=8.3, 1H),5.11 (d, J=12.2, 1H),), 5.18 (d, J=12.2, 1H), 7.31 (m, 5H).

ESI-MS Calcd for C₁₃H₁₇NO₄S: 283.09. Found: 284.1 (M+H)⁺.

Example 114 Synthesis of N-methylsulphonyl-Pro-OH(P1)

A degassed mixture of N-methylsulphonyl-Pro-OBz (P2) (250 mg, 0.88 mmol)and Pd(OH)₂/C (20% Pd, 100 mg, 40% w/w) in IPA:H₂O (26 ml:13 ml), wassaturated with H2 and maintained at 1 atm of hydrogen gas while stirringfor 3 h. Then, the mixture was filtered through a Teflon filter(0.450m), and concentrated under vacuum to yield the title compound (128mg, 75% yield) as a white solid with no further purification.

¹H NMR (300 MHz, CDCl₃) δ 1.94-2.03 (m, 2H), 2.07-2.14 (m, 1H),2.22-2.35 (m, 1H), 2.96 (s, 3H), 3.44 (m, 2H), 4.44 (dd, J₁=3.9, J₂=8.8,2H), 9.10 (m, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 24.96, 31.08, 38.46, 48.06, 60.66, 174.75.

ESI-MS Calcd for C₆H₁₁NO₄S: 193.22. Found: 194.0 (M+H)⁺.

Example 115 Synthesis of [Methylsulphonyl]⁹-aplidine (9MSAPL1)

Following the procedure described for the synthesis of 9ZASHPL2,starting from SAPL2 (10 mg, 10.7 □mol) and N-methylsulphonyl-Pro-OH (10mg, 53 □mol), the title compound (9 mg, 74%) was obtained as a whitesolid after purification by HPLC (Symmetry Prep C18, isocratic ACN/H₂O60:40 (flow: 3 ml/min, 150×7.8 mm, at 270 nm, t_(R)=9 min).

¹H NMR (300 MHz, CDCl₃) δ 0.78-1.00 (m, 24H), 1.20-2.50 (m, 26H), 2.56(s, 3H), 2.93 (m, 1H), 3.04 (s, 3H), 3.06 (s, 3H), 3.08-3.25 (m, 2H),3.28-3.50 (m, 2H), 3.60 (m, 2H), 3.70 (m, 1H), 3.79 (s, 3H), 4.05 (m,2H), 4.17 (m, 1H), 4.60 (m, 2H), 4.81 (m, 2H), 5.10 (m, 1H), 5.19 (d,J=3.4, 1H), 5.33 (m, 1H), 6.84 (d, J=8.3, 2H), 6.86 (m, 1H), 7.07 (d,J=8.3, 2H), 7.09 (m, 1H), 7.82 (d, J=9.2, 1H).

ESI-MS Calcd for C₅₅H₈₇N₇O₁₅S: 1117.60. Found: 1118.7 (M+H)⁺.

Example 116 Synthesis of [Methylsulphonyl]⁸-didemnin A (8MSAPL1)

To a solution of SAPL2 (10 mg, 10.7 □mol) in DCM (200 □l, anh) at 0° C.under Ar, were added DIPEA (3 □l) and methanesulphonyl chloride (0.85□l). The reaction mixture was stirred at 5° C. overnight. DCM (10 ml)was added and the solution was washed successively with aq. KHSO₄ (5 ml,10%), aq. NaHCO₃ (5 ml, sat) and brine (5 ml). The organic phase wasdried (Na₂SO₄), filtered and concentrated to reduce pressure to yieldpure 8MSAPL1 (11 mg, quant) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.83-0.97 (m, 24H), 1.10-1.45 (m, 10H),1.50-1.65 (m, 5H), 1.76-1.82 (m, 2H), 2.00-2.20 (m, 3H), 2.27-2.36 (m,1H), 2.45-2.55 (m, 1H), 2.56 (s, 3H), 2.90 (s, 3H), 3.02 (s, 3H), 3.08(d, J=16.6, 1H), 3.17 (dd, J₁=10.7, J₂=14.1, 1H), 3.37 (m, 1H), 3.60 (m,2H), 3.70 (m, 1H), 3.79 (s, 3H), 3.99-4.11 (m, 3H), 4.49 (m, 1H), 4.59(m, 1H), 4.78 (m, 2H), 5.06 (m, 1H), 5.19 (d, J=3.9, 1H), 6.68 (d,J=8.8, 1H), 6.84 (d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.33 (d, J=9.8,1H), 7.61 (d, J=8.8, 1H).

ESI-MS Calcd for C₅₀H₈₀N₆O₁₄S: 1020.55. Found: 1022.1 (M+H)⁺.

Example 117 Synthesis of [Biotin]⁸-didemnin A (8BISAPL1)

To a solution of HATU (24 mg, 61 □mol), HOAt (8 mg, 63 □mol), SAPL2 (20mg, 21.4 □mol) and d-Biotin (7.8 mg, 32 □mol), in anh. DCM (400 □L) at0° C. under Ar, NMM was added dropwise by syringe. The resulting mixturewas stirred for 2 h at 0° C. and then, at room temperature foradditional 14 h. DCM (10 ml) was added and the solution was washedsuccessively with aq. KHSO₄ (5 ml, 10%), aq. NaHCO₃ (5 ml, sat.) andbrine (5 ml). The organic phase was dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The title compound (18 mg, 72%) wasobtained as a white solid after purification by HPLC (Symmetry Prep C18,gradient ACN/H₂O 60:40-100:0 in 10 min. (flow: 3 ml/min, 150×7.8 mm, at270 nm, t_(R)=6 min).

¹H NMR (300 MHz, CDCl₃) δ 0.78-1.00 (m, 24H), 1.20-1.98 (m, 23H),2.00-2.62 (m, 7H), 2.53 (s, 3H), 2.80-3.00 (m, 2H), 2.86 (s, 3H), 3.16(m, 2H), 3.35 (m, 2H), 3.57 (m, 2H), 3.70 (m, 1H), 3.79 (s, 3H), 4.01(m, 2H), 4.10 (m, 1H), 4.31 (m, 1H), 4.46 (m, 1H), 4.56 (m, 1H), 4.82(m, 2H), 5.00 (m, 1H), 5.14 (m, 2H), 5.67 (s, 1H), 6.23 (s, 1H), 6.84(d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.28 (d, J=8.5, 1H), 7.35 (d,J=7.8, 1H), 8.01 (d, J=8.7, 1H).

ESI-MS Calcd for C₅₉H₉₂N₈O₁₄S: 1168.65. Found: 1169.6 (M+H)⁺.

Example 118 Synthesis of [Phenylurea]⁸-didemnin A (8PUSAPL1)

To a solution of SAPL2 (10 mg, 10.7 □mol) in DCM (200 □l, anh) at 0° C.under Ar, phenyl isocyanate (1.3 □l, 12 □mol) was added and the reactionmixture was stirred at r.t. for 4 hours. DCM (10 ml) was added and thesolution was washed successively with aq. KHSO₄ (5 ml, 10%), aq. NaHCO₃(5 ml, sat) and brine (5 ml). The organic phase was dried (Na₂SO₄),filtered and concentrated to reduced pressure to yield pure 8PUSAPL1 (11mg, 94%) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.85-0.94 (m, 24H), 1.11-1.43 (m, 4H), 1.22(d, J=6.8, 3H), 1.35 (d, J=6.8, 3H), 1.49-1.83 (m, 5H), 1.73 (s, 3H),2.02 (m, 1H), 2.14 (m, 2H), 2.34 (dt, J₁=3.4, J₂=6.8, 1H), 2.54 (s, 3H),2.91 (s, 3H), 3.04 (d, J=16.6, 1H), 3.17 (dd, J₁=11.2, J₂=14.6, 1H),3.36 (dd, J₁=3.9, J₂=14.2, 1H), 3.57 (dd, J₁=4.4, J₂=10.7, 1H),3.59-3.63 (m, 1H), 3.67-3.75 (m, 1H), 3.79 (s, 3H), 3.96-4.10 (m, 2H),4.19 (q, J=6.8, 1H), 4.58 (m, 1H), 4.74 (dd, J₁=3.9, J₂=8.8, 1H),4.78-4.85 (m, 1H), 5.05 (m, 2H), 5.17 (d, J=3.4, 1H), 6.52 (s, 1H), 6.84(d, J=8.3, 2H), 7.03-7.09 (m, 3H), 7.22-7.32 (m, 4H), 7.39 (d, J=8.3,2H), 7.93 (d, J=8.8, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.70, 15.51, 17.16, 18.74, 21.11, 22.41,23.29, 23.98, 24.94, 25.10, 25.32, 26.97, 28.18, 30.36, 31.57, 34.44,36.51, 38.86, 41.64, 45.23, 47.30, 49.80, 50.08, 54.60, 55.50, 56.06,57.51, 62.77, 66.45, 68.17, 70.72, 81.93, 114.36, 120.76, 123.86,129.12, 130.04, 130.57, 138.78, 141.00, 157.78, 158.87, 169.92, 170.68,171.49, 172.43, 173.44, 181.47, 192.38, 204.93, 206.66.

ESI-MS Calcd for C₅₆H₈₃N₇O₁₃: 1161.60. Found: 1062.6 (M+H)⁺.

Example 119 Synthesis of [Pheylthiourea]⁸-didemnin A (8SPSAPL1)

Following the procedure for the synthesis of 8PUSAPL1, starting fromSAPL2 (10 mg, 10.7 □mol) and phenyl thioisocyanate (1.3 □l, 12 □mol),after 15 h of stirring the title compound (11 mg, 93%) was obtained as awhite solid with no further purification.

¹H NMR (300 MHz, CDCl₃) δ 0.84-0.99 (m, 24H), 1.14-1.46 (m, 9H),1.46-1.78 (m, 9H), 1.98-2.19 (m, 3H), 2.34 (m, 1H), 2.54 (s, 3H), 2.91(m, 1H), 3.00 (s, 3H), 3.08-3.21 (m, 1H), 3.36 (dd, J₁=4.4, J₂=14.1,1H), 3.55-3.64 (m, 2H), 3.66-3.74 (m, 1H), 3.79 (s, 3H), 3.96-4.12 (m,2H), 4.21 (q, J=6.8, 1H), 4.59 (t, J=5.3, 1H), 4.75 (dd, J₁=3.4, J₂=8.3,1H), 4.83 (t, J=10.3, 1H), 5.05-5.12 (m, 2H), 5.16 (d, J=3.4, 1H), 6.30(t, J=7.3, 1H), 6.84 (d, J=8.3, 2H), 7.07 (d, J=8.3, 2H), 7.21-7.37 (m,6H), 7.93 (d, J=8.8, 1H), 8.08 (d, J=8.8, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 11.68, 15.28, 15.85, 17.15, 18.73, 21.12,22.73, 23.66, 24.01, 25.13, 25.33, 26.62, 28.16, 29.92, 31.53, 32.42,32.90, 34.41, 36.77, 38.86, 41.69, 47.28, 50.12, 55.50, 56.48, 57.51,59.87, 66.47, 70.75, 81.91, 114.37, 125.25, 125.95, 126.57, 127.54,129.12, 129.77, 130.06, 130.57, 135.43, 158.88, 168.54, 169.90, 170.70,171.49, 172.38, 190.41.

ESI-MS Calcd for C₅₆H₈₃N₇O₁₂S: 1077.58. Found: 1078.5 (M+H)⁺.

Example 120 Synthesis of [Butylurea]⁸-didemnin A (8BUSAPL1)

Following the procedure for the synthesis of 8PUSAPL1, starting fromSAPL2 (10 mg, 10.7 □mol) and butyl thioisocyanate (1.4 □l, 12 □mol),after 4 h of stirring the title compound (9 mg, 78%) was obtained as awhite solid with no further purification.

¹H NMR (300 MHz, CDCl₃) δ 0.85-0.94 (m, 24H), 1.10-1.80 (m, 24H), 2.03(m, 1H), 2.13 (m, 2H), 2.33 (m, 1H), 2.55 (s, 3H), 2.72 (s, 3H), 3.02(d, J=16.1, 1H), 3.17 (dd, J₁=11.2, J₂=14.2, 1H), 3.23-3.40 (m, 3H),3.62-3.78 (m, 3H), 3.79 (s, 3H), 4.03 (m, 2H), 4.19 (q, J=6.8, 1H), 4.58(m, 2H), 4.71 (dd, J₁=3.4, J₂=8.3, 1H), 4.82 (m, 1H), 5.00 (m, 2H), 5.16(d, J=3.4, 1H), 5.22-5.28 (m, 2H), 6.85 (d, J=8.8, 2H), 7.08 (d, J=8.3,2H), 7.21-7.29 (m, 1H), 7.96 (d, J=9.2, 1H).

ESI-MS Calcd for C₅₄H₈₇N₇O₁₃: 1041.64. Found: 1042.7 (M+H)⁺.

Example 121 Synthesis of [Butylthiourea]⁸-didemnin A (8BTSAPL1)

Following the procedure for the synthesis of 8PUSAPL1, starting fromSAPL2 (10 mg, 10.7 □mol) and butyl thioisocyanate (1.5 □l, 12 □mol),after 15 h of reaction the title compound (10 mg, 86%) was obtained as awhite solid with no further purification.

¹H NMR (300 MHz, CDCl₃) δ 0.86-0.97 (m, 24H), 1.14-1.78 (m, 27H),1.99-2.14 (m, 3H), 2.34 (m, 1H), 2.55 (s, 3H), 2.88 (s, 3H), 3.01 (d,J=16.6, 1H), 3.16 (dd, J₁=11.2, J₂=14.6, 1H), 3.36 (dd, J₁=4.4, J₂=14.2,1H), 3.49-3.74 (m, 3H), 3.79 (s, 3H), 4.02 (d, J=6.8, 2H), 4.21 (q,J=6.8, 1H), 4.58 (m, 1H), 4.70 (dd, J₁=2.9, J₂=8.3, 1H), 4.82 (t,J=10.3, 1H), 5.07-5.12 (m, 2H), 5.09 (d, J=3.9, 1H), 5.60 (m, 1H), 6.36(dd, J₁=5.4. J₂=8.3, 1H), 6.84 (d, J=8.8, 2H), 7.08 (d, J=8.8, 2H), 7.84(d, J=8.3, 1H), 7.91 (d, J=9.3, 1H).

ESI-MS Calcd for C₅₄H₈₇N₇O₁₂S: 1057.61. Found: 1080.7 (M+Na)⁺.

1-51. (canceled)
 52. A method of making a didemnin fragment, the methodcomprising coupling a first reactant having the structure

and a second reactant having the structure

to yield a didemnin fragment having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or
 1. 53. The method of claim 52,further comprising deprotection the didemnin fragment to yield adidemnin fragment having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or
 1. 54. A method of making adidemnin fragment comprising the cyclizing the fragment of claim 53 toyield a didemnin analog having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or
 1. 55. The method of claim 54,further comprising hydrolyzing the didemnin analog to yield a didemninanalog having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or
 1. 56. A method of making adidemnin analog, the method comprising coupling a first reactant havingthe structure

and a second reactant having the structure

to yield a didemnin analog having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or
 1. 57. The method of claim 56,further comprising deprotection the didemnin fragment to yield adidemnin fragment having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or
 1. 58. A method of making adidemnin fragment comprising the coupling of the fragment having thestructure

and a second reactant having the structure

to yield a didemnin fragment having the structure


59. The method of claim 58, further comprising deprotection the didemninfragment to yield a didemnin fragment having the structure


60. A method of making a didemnin analog comprising the coupling of thedidemnin analog in claim 57 with the fragment in the claim 59, to yieldthe didemnin analog having the structure

wherein X is selected from the group consisting of —O—, and —NH—; whereY is —(COCHCH₃)_(n)CO—; where n is 0 or
 1. 61. (canceled)
 62. A methodof making a didemnin analog comprising the coupling of the didemninanalog having the structure

and the fragment having the structure

to yield the didemnin analog having the structure


63. The method of claim 62, further comprising deprotection the didemninanalog to yield a didemnin fragment having the structure

64-67. (canceled)
 68. A method of making a didemnin analog comprisingthe coupling of the didemnin analog having the structure

and the fragment having the structure

to yield the didemnin analog having the structure

wherein R is SO₂Me, and Z-Nva.
 69. The method of claim 68, furthercomprising deprotection the didemnin analog to yield a didemnin analoghaving the structure

wherein R₂ is Nva. 70-75. (canceled)
 76. A compound of formula:

wherein: X is independently —C(R)₂—, —O—, —S—, or —NR—, in which R isindependently H or an organic group selected from an alkyl group, analkenyl group, an aryl group, an aralkyl group, and their substitutedderivatives substituted with one or more of a heterocyclic group, analkoxy group, an hydroxy group, a mercapto group, an optionallyprotected amino group, a guanidino group, or a halogen group; Y is CO or—COCHCH₃CO—; R₄ is H or an organic group selected from a group RSO₂—, anamido group RCONH— or an acyl group RCO— where R is as defined, an alkylgroup, an alkenyl group, an aryl group, an aralkyl group, andsubstituted derivatives substituted with one or more of a heterocyclicgroup, an alkoxy group, an hydroxy group, a mercapto group, anoptionally protected amino group, a guanidino group, or a halogen group;X₁ is O or S; and when Y is CO, then R₃ is independently H or an organicgroup selected from an alkyl group, an alkenyl group, an aryl group, anaralkyl group, a group RSO₂— or an acyl group RCO—, where R is an alkylgroup, an alkenyl group, an aryl group, an aralkyl group, andsubstituted derivatives substituted with one or more of a mercaptogroup, an optionally protected amino group, a guanidino group, or ahalogen group; when Y is —COCHCH₃CO—, then R₃ is RSO₂— where R is alkyl,or R₃ is an alkenyl group, an aralkyl group, and substituted derivativessubstituted with one or more of a carbonyl group, an hydroxy group, amercapto group, an optionally protected amino group, a guanidino group,or a halogen group; and pharmaceutically acceptable salts thereof.
 77. Acompound according to claim 76, wherein X is —NR—, in which R is asdefined.
 78. A compound according to claim 77, wherein X is —NH— or—NMe—.
 79. A compound according to claim 78, wherein X is —NH—.
 80. Acompound according to claim 76, wherein X is —O—.
 81. A compoundaccording to claim 76, wherein Y is —CO—.
 82. A compound according toclaim 76, wherein X is —NH— or —O— and Y is —COCHCH₃CO— or —CO—.
 83. Acompound according to claim 76, wherein F4 is methyl.
 84. A compoundaccording to claim 76, which is selected from:3-[Hiv]-9-[Isobutyryl]-aplidine, 3-[Val]-9-[Isobutyryl]-aplidine,3-[Hiv]-9-[Ala]-aplidine, 3-[Hiv]-9-[Nva-Pro]-aplidine,3-[Hiv]-9-[Z-ala]-aplidine, 3-[Hiv]-9-[Z-Nva-Pro]-aplidine,3-[Hiv]-9-[Boc-Ala]-aplidine, or 3-[Hiv]-8-[Pro]-didemnin A, or apharmaceutically acceptable salts thereof.
 85. A method of treating of atumour which comprises administering a compound according to claim 76.86. A pharmaceutical preparation which comprises a compound according toclaim 76 and a pharmaceutically acceptable carrier.